Materials and Methods Involving Hybrid Vascular Endothelial Growth Factor DNAs and Proteins

ABSTRACT

The present invention provides polypeptides that bind cellular receptors for vascular endothelial growth factor polypeptides; polynucleotides encoding such polypeptides; compositions comprising the polypeptides and polynucleotides; and methods and uses involving the foregoing. Some polypeptides of the invention exhibit unique receptor binding profiles compared to known, naturally occurring vascular endothelial growth factors.

The present application is a continuation application of U.S. patentapplication Ser. No. 11/064,774, filed Feb. 24, 2005, which is adivisional application of U.S. patent application Ser. No. 09/795,006,filed Feb. 26, 2001, now U.S. Pat. No. 6,965,010, which claims thebenefit of priority to U.S. Provisional Patent Application No.60/205,331 filed May 18, 2000 and U.S. Provisional Patent ApplicationNo. 60/185,205 filed Feb. 25, 2000. The entire text and drawing of eachof the priority applications is specifically incorporated herein byreference, without prejudice or disclaimer.

BACKGROUND OF THE INVENTION

The PDGF proteins and their receptors (PDGFRs) are involved inregulation of cell proliferation, survival and migration of several celltypes. The VEGF proteins and their receptors (VEGFRs) play importantroles in both vasculogenesis, the development of the embryonicvasculature from early differentiating endothelial cells, andangiogenesis, the process of forming new blood vessels from pre-existingones [Risau et al., Dev Biol 125:441-450 (1988); Zachary, Intl J BiochemCell Bio 30:1169-1174 (1998); Neufeld et al., FASEB J 13:9-22 (1999);Ferrara, J Mol Med 77:527-543 (1999)]. Both processes depend on thetightly controlled endothelial cell proliferation, migration,differentiation, and survival. Dysfunction of the endothelial cellregulatory system is a key feature of cancer and several diseasesassociated with abnormal angiogenesis, such as proliferativeretinopathies, age-related muscular degeneration, rheumatoid arthritis,and psoriasis. Understanding of the specific biological function of thekey players involved in regulating endothelial cells will lead to moreeffective therapeutic applications to treat such diseases [Zachary, IntlJ Biochem Cell Bio 30:1169-1174 (1998); Neufeld et al., FASEB J 13:9-22(1999); Ferrara, J Mol Med 77:527-543 (1999)].

The PDGF/VEGF Family

The PDGF/VEGF family of growth factors includes at least the followingmembers: PDGF-A (see e.g., GenBank Acc. No. X06374), PDGF-B (see e.g.,GenBank Acc. No. M12783), VEGF (see e.g., GenBank Acc. No. Q16889referred to herein for clarity as VEGF-A or by particular isoform), PlGF(see e.g., GenBank Acc. No. X54936 placental growth factor), VEGF-B (seee.g., GenBank Acc. No. U48801; also known as VEGF-related factor (VRF)),VEGF-C (see e.g., GenBank Acc. No. X94216; also known as VEGF relatedprotein (VRP)), VEGF-D (also known as c-fos-induced growth factor(FIGF); see e.g., Genbank Acc. No. AJ000185), VEGF-E (also known as NZ7VEGF or OV NZ7; see e.g., GenBank Acc. No. S67522), NZ2 VEGF (also knownas OV NZ2; see e.g., GenBank Acc. No. S67520), D1701 VEGF-like protein(see e.g., GenBank Acc. No. AF106020; Meyer et al., EMBO J 18:363-374),and NZ10 VEGF-like protein (described in International PatentApplication PCT/US99/25869) [Stacker and Achen, Growth Factors 17:1-11(1999); Neufeld et al., FASEB J 13:9-22 (1999); Ferrara, J Mol Med77:527-543 (1999)].

Members of the PDGF/VEGF family are characterized by a number ofstructural motifs including a conserved PDGF motif defined by thesequence: P-[PS]-C-V-X(3)-R-C-[GSTA]-G-C-C (SEQ ID NO: 1200). Thebrackets indicate that this position within the polypeptide can be anyone of the amino acids contained within the brackets. The numbercontained within the parentheses indicates the number of amino acidsthat separate the “V” and “R” residues. This conserved motif fallswithin a large domain of 70-150 amino acids defined in part by eighthighly conserved cysteine residues that form inter- and intramoleculardisulfide bonds. This domain forms a cysteine knot motif composed of twodisulfide bonds which form a covalently linked ring structure betweentwo adjacent B strands, and a third disulfide bond that penetrates thering [see for example, FIG. 1 in Muller et al., Structure 5:1325-1338(1997)], similar to that found in other cysteine knot growth factors,e.g., transforming growth factor-β (TGF-β). The amino acid sequence ofall known PDGF/VEGF proteins, with the exception of VEGF-E, contains thePDGF domain. The PDGF/VEGF family proteins are predominantly secretedglycoproteins that form either disulfide-linked or non-covalently boundhomo- or heterodimers whose subunits are arranged in an anti-parallelmanner [Stacker and Achen, Growth Factors 17:1-11 (1999); Muller et al.,Structure 5:1325-1338 (1997)].

The PDGF Subfamily

The PDGFs regulate cell proliferation, cell survival and chemotaxis ofmany cell types in vitro (reviewed in [Heldin et al., Biochimica etBiophysica Acta 1378:F79-113 (1998)]. The two chains that make up PDGF,PDGF-A and PDGF-B, can homo- or heterodimerize producing three differentisoforms: PDGF-AA, PDGF-AB, or PDGF-BB. PDGF-A is only able to bind thePDGF α-receptor (PDGFR-α), whereas PDGF-B can bind both the PDGF-α and asecond PDGF receptor (PDGF-β). In vivo, the PDGF proteins exert theireffects in a paracrine manner since they often are expressed inepithelial (PDGF-A) or endothelial (PDGF-B) cells in close apposition tothe PDGF receptor-expressing mesenchyme (reviewed in Ataliotis et al.,Int Rev Cytology 172:95-127 (1997)]. Overexpression of the PDGFs hasbeen observed in several pathological conditions, includingmalignancies, atherosclerosis, and fibroproliferative diseases. In tumorcells and cell lines grown in vitro, co-expression of the PDGFs and PDGFreceptors generates autocrine loops, which are important for cellulartransformation [Betsholtz et al., Cell 39:447-57 (1984); Keating et al.,Science 239:914-6 (1988)].

The importance of the PDGFs as regulators of cell proliferation and cellsurvival is well illustrated by recent gene targeting studies in mice.Homozygous null mutations for either PDGF-A or PDGF-B are lethal inmice. Approximately 50% of the homozygous PDGF-A deficient mice have anearly lethal phenotype, while the surviving animals have a complexpostnatal phenotype with lung emphysema due to improper alveolar septumformation, and a dermal phenotype characterized by thin dermis,misshapen hair follicles, and thin hair. PDGF-A is also required fornormal development of oligodendrocytes and subsequent myelination of thecentral nervous system. The PDGF-B deficient mice develop renal,hematological and cardiovascular abnormalities; where the renal andcardiovascular defects, at least in part, are due to the lack of properrecruitment of mural cells (vascular smooth muscle cells, pericytes ormesangial cells) to blood vessels.

The VEGF Subfamily

The VEGF subfamily is composed of PDGF/VEGF members which share a VEGFhomology domain (VHD) characterized by the sequence:C-X(22-24)-P-[PSR]-C-V-X(3)-R-C-[GSTA]-G-C-C-X(6)-C-X(32-41)-C (SEQ IDNO: 1201). The VHD domain, determined through analysis of the VEGFsubfamily members, comprises the PDGF motif but is more specific.

VEGF-A was originally purified from several sources on the basis of itsmitogenic activity toward endothelial cells, and also by its ability toinduce microvascular permeability, hence it is also called vascularpermeability factor (VPF). VEGF-A has subsequently been shown to inducea number of biological processes including the mobilization ofintracellular calcium, the induction of plasminogen activator andplasminogen activator inhibitor-1 synthesis, promotion of monocytemigration in vitro, induction of antiapoptotic protein expression inhuman endothelial cells, induction of fenestrations in endothelialcells, promotion of cell adhesion molecule expression in endothelialcells and induction of nitric oxide mediated vasodilation andhypotension [Ferrara, J Mol Med 77: 527-543 (1999); Neufeld et al.,FASEB J 13: 9-22 (1999); Zachary, Intl J Biochem Cell Bio 30: 1169-1174(1998)].

VEGF-A is a secreted, disulfide-linked homodimeric glycoprotein composedof 23 kD subunits. Five human VEGF-A isoforms of 121, 145, 165, 189 or206 amino acids in length (VEGF₁₂₁₋₂₀₆), encoded by distinct mRNA splicevariants, have been described, all of which are capable of stimulatingmitogenesis in endothelial cells. However, each isoform differs inbiological activity, receptor specificity, and affinity for cellsurface- and extracellular matrix-associated heparan-sulfateproteoglycans, which behave as low affinity receptors for VEGF-A.VEGF₁₂₁ does not bind to either heparin or heparan-sulfate; VEGF₁₄₅ andVEGF₁₆₅ (GenBank Acc. No. M32977) are both capable of binding toheparin; and VEGF₁₈₉ and VEGF₂₀₆ show the strongest affinity for heparinand heparan-sulfates. VEGF₁₂₁, VEGF₁₄₅, and VEGF₁₆₅ are secreted in asoluble form, although most of VEGF₁₆₅ is confined to cell surface andextracellular matrix proteoglycans, whereas VEGF₁₈₉ and VEGF₂₀₆ remainassociated with extracellular matrix. Both VEGF₁₈₉ and VEGF₂₀₆ can bereleased by treatment with heparin or heparinase, indicating that theseisoforms are bound to extracellular matrix via proteoglycans. Cell-boundVEGF₁₈₉ can also be cleaved by proteases such as plasmin, resulting inrelease of an active soluble VEGF₁₁₀. Most tissues that express VEGF areobserved to express several VEGF isoforms simultaneously, althoughVEGF₁₂, and VEGF₁₆₅ are the predominant forms, whereas VEGF₂₀₆ is rarelydetected [Ferrara, J Mol Med 77:527-543 (1999)]. VEGF145 differs in thatit is primarily expressed in cells derived from reproductive organs[Neufeld et al., FASEB J 13:9-22 (1999)].

The pattern of VEGF-A expression suggests its involvement in thedevelopment and maintenance of the normal vascular system, and inangiogenesis associated with tumor growth and other pathologicalconditions such as rheumatoid arthritis. VEGF-A is expressed inembryonic tissues associated with the developing vascular system, and issecreted by numerous tumor cell lines. Analysis of mice in which VEGF-Awas knocked out by targeted gene disruption indicate that VEGF-A iscritical for survival, and that the development of the cardiovascularsystem is highly sensitive to VEGF-A concentration gradients. Micelacking a single copy of VEGF-A die between day 11 and 12 of gestation.These embryos show impaired growth and several developmentalabnormalities including defects in the developing cardiovasculature.VEGF-A is also required post-natally for growth, organ development,regulation of growth plate morphogenesis and endochondral boneformation. The requirement for VEGF-A decreases with age, especiallyafter the fourth postnatal week. In mature animals, VEGF-A is requiredprimarily for active angiogenesis in processes such as wound healing andthe development of the corpus luteum. [Neufeld et al., FASEB J 13:9-22(1999); Ferrara, J Mol Med 77:527-543 (1999)]. VEGF-A expression isinfluenced primarily by hypoxia and a number of hormones and cytokinesincluding epidermal growth factor (EGF), TGF-β, and variousinterleukins. Regulation occurs transcriptionally and alsopost-transcriptionally such as by increased mRNA stability [Ferrara, JMol Med 77:527-543 (1999)].

PlGF, a second member of the VEGF subfamily, is generally a poorstimulator of angiogenesis and endothelial cell proliferation incomparison to VEGF-A, and the in vivo role of PlGF is not wellunderstood. Three isoforms of PlGF produced by alternative mRNA splicinghave been described [Hauser et al., Growth Factors 9:259-268 (1993);Maglione et al., Oncogene 8:925-931 (1993)]. PlGF forms bothdisulfide-liked homodimers and heterodimers with VEGF-A. The PlGF-VEGF-Aheterodimers are more effective at inducing endothelial cellproliferation and angiogenesis than PlGF homodimers. PlGF is primarilyexpressed in the placenta, and is also co-expressed with VEGF-A duringearly embryogenesis in the trophoblastic giant cells of the parietalyolk sac [Stacker and Achen, Growth Factors 17:1-11 (1999)].

VEGF-B, described in detail in International Patent Publication No. WO96/26736 and U.S. Pat. Nos. 5,840,693 and 5,607,918, sharesapproximately 44% amino acid identity with VEGF-A. Although thebiological functions of VEGF-B in vivo remain incompletely understood,it has been shown to have angiogenic properties, and may also beinvolved in cell adhesion and migration, and in regulating thedegradation of extracellular matrix. It is expressed as two isoforms of167 and 186 amino acid residues generated by alternative splicing.VEGF-B167 is associated with the cell surface or extracellular matrixvia a heparin-binding domain, whereas VEGF-B186 is secreted. BothVEGF-B167 and VEGF-B186 can form disulfide-linked homodimers orheterodimers with VEGF-A. The association to the cell surface ofVEGF165-VEGF-B167 heterodimers appears to be determined by the VEGF-Bcomponent, suggesting that heterodimerization may be important forsequestering VEGF-A. VEGF-B is expressed primarily in embryonic andadult cardiac and skeletal muscle tissues [Joukov et al., J Cell Physiol173:211-215 (1997); Stacker and Achen, Growth Factors 17:1-11 (1999)].Mice lacking VEGF-B survive but have smaller hearts, dysfunctionalcoronary vasculature, and exhibit impaired recovery from cardiacischemia [Bellomo et al., Circ Res 2000; E29-E35].

A fourth member of the VEGF subfamily, VEGF-C, comprises a VHD that isapproximately 30% identical at the amino acid level to VEGF-A. VEGF-C isoriginally expressed as a larger precursor protein, prepro-VEGF-C,having extensive amino- and carboxy-terminal peptide sequences flankingthe VHD, with the C-terminal peptide containing tandemly repeatedcysteine residues in a motif typical of Balbiani ring 3 protein.Prepro-VEGF-C undergoes extensive proteolytic maturation involving thesuccessive cleavage of a signal peptide, the C-terminal pro-peptide, andthe N-terminal pro-peptide. Secreted VEGF-C protein consists of anon-covalently-linked homodimer, in which each monomer contains the VHD.The intermediate forms of VEGF-C produced by partial proteolyticprocessing show increasing affinity for the VEGFR-3 receptor, and themature protein is also able to bind to the VEGFR-2 receptor. [Joikov etal., EMBO J., 16:(13):3898-3911 (1997).] It has also been demonstratedthat a mutant VEGF-C, in which a single cysteine at position 156 iseither substituted by another amino acid or deleted, loses the abilityto bind VEGFR-2 but remains capable of binding and activating VEGFR-3[International Patent Publication No. WO 98/33917]. In mouse embryos,VEGF-C mRNA is expressed primarily in the allantois, jugular area, andthe metanephros. [Joukov et al., J Cell Physiol 173:211-215 (1997)].VEGF-C is involved in the regulation of lymphatic angiogenesis: whenVEGF-C was overexpressed in the skin of transgenic mice, a hyperplasticlymphatic vessel network was observed, suggesting that VEGF-C induceslymphatic growth [Jeltsch et al., Science, 276:1423-1425 (1997)].Continued expression of VEGF-C in the adult also indicates a role inmaintenance of differentiated lymphatic endothelium [Ferrara, J Mol Med77:527-543 (1999)]. VEGF-C also shows angiogenic properties: it canstimulate migration of bovine capillary endothelial (BCE) cells incollagen and promote growth of human endothelial cells [see, e.g.,International Patent Publication No. WO 98/33917, incorporated herein byreference].

VEGF-D is structurally and functionally most closely related to VEGF-C[see International Patent Publ. No. WO 98/07832, incorporated herein byreference]. Like VEGF-C, VEGF-D is initially expressed as aprepro-peptide that undergoes N-terminal and C-terminal proteolyticprocessing, and forms non-covalently linked dimers. VEGF-D stimulatesmitogenic responses in endothelial cells in vitro. During embryogenesis,VEGF-D is expressed in a complex temporal and spatial pattern, and itsexpression persists in the heart, lung, and skeletal muscles in adults.Isolation of a biologically active fragment of VEGF-D designatedVEGF-DΔNΔC, is described in International Patent Publication No. WO98/07832, incorporated herein by reference. VEGF-DΔNΔC consists of aminoacid residues 93 to 201 of VEGF-D linked to the affinity tag peptideFLAG®.

Four additional members of the VEGF subfamily have been identified inpoxviruses, which infect humans, sheep and goats. The orf virus-encodedVEGF-E and NZ2 VEGF are potent mitogens and permeability enhancingfactors. Both show approximately 25% amino acid identity to mammalianVEGF-A, and are expressed as disulfide-liked homodimers. Infection bythese viruses is characterized by pustular dermititis which may involveendothelial cell proliferation and vascular permeability induced bythese viral VEGF proteins. [Ferrara, J Mol Med 77:527-543 (1999);Stacker and Achen, Growth Factors 17:1-11 (1999)]. VEGF-like proteinshave also been identified from two additional strains of the orf virus,D1701 [GenBank Acc. No. AF106020; described in Meyer et al., EMBO J18:363-374 (1999)] and NZ10 [described in International PatentApplication PCT/US99/25869, incorporated herein by reference]. Theseviral VEGF-like proteins have been shown to bind VEGFR-2 present on hostendothelium, and this binding is important for development of infectionand viral induction of angiogenesis [Meyer et al., EMBO J 18:363-374(1999); International Patent Application PCT/US99/25869].

PDGF/VEGF Receptors

Seven cell surface receptors that interact with PDGF/VEGF family membershave been identified. These include PDGFR-α (see e.g., GenBank Acc. No.NM006206), PDGFR-β (see e.g., GenBank Acc. No. NM002609), VEGFR-1/Flt-1(fms-like tyrosine kinase-1; GenBank Acc. No. X51602; De Vries et al.,Science 255:989-991 (1992)); VEGFR-2/KDR/Flk-1 (kinase insert domaincontaining receptor/fetal liver kinase-1; GenBank Acc. Nos. X59397(Flk-1) and L04947 (KDR); Terman et al., Biochem Biophys Res Comm187:1579-1586 (1992); Matthews et al., Proc Natl Acad Sci USA88:9026-9030 (1991)); VEGFR-3/Flt4 (fms-like tyrosine kinase 4; U.S.Pat. No. 5,776,755 and GenBank Acc. No. X68203 and S66407; Pajusola etal., Oncogene 9:3545-3555 (1994)), neuropilin-1 (Gen Bank Acc. No.NM003873), and neuropilin-2 (Gen Bank Acc. No. NM003872). The two PDGFreceptors mediate signaling of PDGFs as described above. VEGF121,VEGF165, VEGF-B, PlGF-1 and PlGF-2 bind VEGF-R1; VEGF121, VEGF145,VEGF165, VEGF-C, VEGF-D, VEGF-E, and NZ2 VEGF bind VEGF-R2; VEGF-C andVEGF-D bind VEGFR-3; VEGF165, PlGF-2, and NZ2 VEGF bind neuropilin-1;and VEGF165 binds neuropilin-2. [Neufeld et al., FASEB J 13:9-22 (1999);Stacker and Achen, Growth Factors 17:1-11 (1999); Ortega et al., FronBiosci 4:141-152 (1999); Zachary, Intl J Biochem Cell Bio 30:1169-1174(1998); Petrova et al., Exp Cell Res 253:117-130 (1999)].

The PDGF receptors are protein tyrosine kinase receptors (PTKs) thatcontain five immunoglobulin-like loops in their extracellular domains.VEGFR-1, VEGFR-2, and VEGFR-3 comprise a subgroup of the PDGF subfamilyof PTKs, distinguished by the presence of seven Ig domains in theirextracellular domain and a split kinase domain in the cytoplasmicregion. Both neuropilin-1 and neuropilin-2 are non-PTK VEGF receptors.NP-1 has an extracellular portion includes a MAM domain; regions ofhomology to coagulation factors V and VIII, MFGPs and the DDR tyrosinekinase; and two CUB-like domains.

Several of the VEGF receptors are expressed as more than one isoform. Asoluble isoform of VEGFR-1 lacking the seventh Ig-like loop,transmembrane domain, and the cytoplasmic region is expressed in humanumbilical vein endothelial cells. This VEGFR-1 isoform binds VEGF-A withhigh affinity and is capable of preventing VEGF-A-induced mitogenicresponses [Ferrara, J Mol Med 77:527-543 (1999); Zachary, Intl J BiochemCell Bio 30:1169-1174 (1998)]. A C-terminal truncated from of VEGFR-2has also been reported [Zachary, Intl J Biochem Cell Bio 30:1169-1174(1998)]. In humans, there are two isoforms of the VEGFR-3 protein whichdiffer in the length of their C-terminal ends. Studies suggest that thelonger isoform is responsible for most of the biological properties ofVEGFR-3.

The receptors for the PDGFs, PDGF α-receptor (PDGFR-α) and theβ-receptor (PDGFR-β), are expressed by many in vitro grown cell lines,and they are mainly expressed by mesenchymal cells in vivo (reviewed in[Raines et al., Peptide growth factors and their receptors, Heidelberg,Springer-Verlag (1990)]. As mentioned above, PDGF-B binds both PDGFRs,while PDGF-A selectively binds PDGFR-α.

Gene targeting studies in mice have revealed distinct physiologicalroles for the PDGF receptors despite the overlapping ligandspecificities of the PDGFRs [Rosenkranz et al., Growth Factors 16:201-16(1999)]. Homozygous null mutations for either of the two PDGF receptorsare lethal. PDGFR-α deficient mice die during embryogenesis at e10, andshow incomplete cephalic closure, impaired neural crest development,cardiovascular defects, skeletal defects, and odemas. The PDGFR-βdeficient mice develop similar phenotypes to animals deficient inPDGF-B, that are characterized by renal, hematological andcardiovascular abnormalities; where the renal and cardiovasculardefects, at least in part, are due to the lack of proper recruitment ofmural cells (vascular smooth muscle cells, pericytes or mesangial cells)to blood vessels.

The expression of VEGFR-1 occurs mainly in vascular endothelial cells,although some may be present on monocytes, trophoblast cells, and renalmesangial cells [Neufeld et al., FASEB J 13:9-22 (1999)]. High levels ofVEGFR-1 mRNA are also detected in adult organs, suggesting that VEGFR-1has a function in quiescent endothelium of mature vessels not related tocell growth. VEGFR-1−/− mice die in utero between day 8.5 and 9.5.Although endothelial cells developed in these animals, the formation offunctional blood vessels was severely impaired, suggesting that VEGFR-1may be involved in cell-cell or cell-matrix interactions associated withcell migration. Recently, it has been demonstrated that mice expressinga mutated VEGFR-1 in which only the tyrosine kinase domain was missingshow normal angiogenesis and survival, suggesting that the signalingcapability of VEGFR-1 is not essential. [Neufeld et al., FASEB J 13:9-22(1999); Ferrara, J Mol Med 77:527-543 (1999)].

VEGFR-2 expression is similar to that of VEGFR-1 in that it is broadlyexpressed in the vascular endothelium, but it is also present inhematopoietic stem cells, megakaryocytes, and retinal progenitor cells[Neufeld et al., FASEB J 13:9-22 (1999)]. Although the expressionpattern of VEGFR-1 and VEGFR-2 overlap extensively, evidence suggeststhat, in most cell types, VEGFR-2 is the major receptor through whichmost of the VEGFs exert their biological activities. Examination ofmouse embryos deficient in VEGFR-2 further indicate that this receptoris required for both endothelial cell differentiation and thedevelopment of hematopoietic cells [Joukov et al., J Cell Physiol173:211-215 (1997)].

VEGFR-3 is expressed broadly in endothelial cells during earlyembryogenesis. During later stages of development, the expression ofVEGFR-3 becomes restricted to developing lymphatic vessels [Kaipainen,A., et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)]. Inadults, the lymphatic endothelia and some high endothelial venulesexpress VEGFR-3, and increased expression occurs in lymphatic sinuses inmetastatic lymph nodes and in lymphangioma. VEGFR-3 is also expressed ina subset of CD34⁺ hematopoietic cells which may mediate the myelopoieticactivity of VEGF-C demonstrated by overexpression studies [WO 98/33917].Targeted disruption of the VEGFR-3 gene in mouse embryos leads tofailure of the remodeling of the primary vascular network, and deathafter embryonic day 9.5 [Dumont et al., Science, 282: 946-949 (1998)].These studies suggest an essential role for VEGFR-3 in the developmentof the embryonic vasculature, and also during lymphangiogenesis.

Structural analyses of the VEGF receptors indicate that the VEGF-Abinding site on VEGFR-1 and VEGFR-2 is located in the second and thirdIg-like loops. Similarly, the VEGF-C and VEGF-D binding sites on VEGFR-2and VEGFR-3 are also contained within the second Ig-loop [Taipale etal., Curr Top Microbiol Immunol 237:85-96 (1999)]. The second Ig-likeloop also confers ligand specificity as shown by domain swappingexperiments [Ferrara, J Mol Med 77:527-543 (1999)]. Receptor-ligandstudies indicate that dimers formed by the VEGF family proteins arecapable of binding two VEGF receptor molecules, thereby dimerizing VEGFreceptors. The fourth Ig-like loop on VEGFR-1, and also possibly onVEGFR-2, acts as the receptor dimerization domain that links tworeceptor molecules upon binding of the receptors to a ligand dimer[Ferrara, J Mol Med 77:527-543 (1999)]. Although the regions of VEGF-Athat bind VEGFR-1 and VEGFR-2 overlap to a large extent, studies haverevealed two separate domains within VEGF-A that interact with eitherVEGFR-1 or VEGFR-2, as well as specific amino acid residues within thesedomains that are critical for ligand-receptor interactions. Mutationswithin either VEGF receptor-specific domain that specifically preventbinding to one particular VEGF receptor have also been recovered[Neufeld et al., FASEB J 13:9-22 (1999)].

VEGFR-1 and VEGFR-2 are structurally similar, share common ligands(VEGF₁₂₁ and VEGF₁₆₅), and exhibit similar expression patterns duringdevelopment. However, the signals mediated through VEGFR-1 and VEGFR-2by the same ligand appear to be slightly different. VEGFR-2 has beenshown to undergo autophosphorylation in response to VEGF-A, butphosphorylation of VEGFR-1 under identical conditions was barelydetectable. VEGFR-2 mediated signals cause striking changes in themorphology, actin reorganization, and membrane ruffling of porcineaortic endothelial cells recombinantly overexpressing this receptor. Inthese cells, VEGFR-2 also mediated ligand-induced chemotaxis andmitogenicity; whereas VEGFR-1-transfected cells lacked mitogenicresponses to VEGF-A. Mutations in VEGF-A that disrupt binding to VEGFR-2fail to induce proliferation of endothelial cells, whereas VEGF-Amutants that are deficient in binding VEGFR-1 are still capable ofpromoting endothelial proliferation. Similarly, VEGF stimulation ofcells expressing only VEGFR-2 leads to a mitogenic response whereascomparable stimulation of cells expressing only VEGFR-1 also results incell migration, but does not induce cell proliferation. In addition,phosphoproteins co-precipitating with VEGFR-1 and VEGFR-2 are distinct,suggesting that different signaling molecules interact withreceptor-specific intracellular sequences.

The emerging hypothesis is that the primary function of VEGFR-1 inangiogenesis may be to negatively regulate the activity of VEGF-A bybinding it and thus preventing its interaction with VEGFR-2, whereasVEGFR-2 is thought to be the main transducer of VEGF-A signals inendothelial cells. In support of this hypothesis, mice deficient inVEGFR-1 die as embryos while mice expressing a VEGFR-1 receptor capableof binding VEGF-A but lacking the tyrosine kinase domain survive and donot exhibit abnormal embryonic development or angiogenesis. In addition,analyses of VEGF-A mutants that bind only VEGFR-2 show that they retainthe ability to induce mitogenic responses in endothelial cells. However,VEGF-mediated migration of monocytes is dependent on VEGFR-1, indicatingthat signaling through this receptor is important for at least onebiological function. In addition, the ability of VEGF-A to prevent thematuration of dendritic cells is also associated with VEGFR-1 signaling,suggesting that VEGFR-1 may function in cell types other thanendothelial cells. [Ferrara, J Mol Med 77:527-543 (1999); Zachary, IntlJ Biochem Cell Bio 30:1169-1174 (1998)].

Neuropilin-1 was originally cloned as a receptor for thecollapsin/semaphorin family of proteins involved in axon guidance[Stacker and Achen, Growth Factors 17:1-11 (1999)]. It is expressed inboth endothelia and specific subsets of neurons during embryogenesis,and it thought to be involved in coordinating the developing neuronaland vascular system. Although activation of neuropilin-1 does not appearto elicit biological responses in the absence of the VEGF familytyrosine-kinase receptors, their presence on cells leads to moreefficient binding of VEGF165 and VEGFR-2 mediated responses. [Neufeld etal., FASEB J 13:9-22 (1999)] Mice lacking neuropilin-1 showabnormalities in the developing embryonic cardiovascular system.[Neufeld et al., FASEB J 13:9-22 (1999)]

Neuropilin-2 was identified by expression cloning and is acollapsin/semaphorin receptor closely related to neuropilin-1.Neuropilin-2 is an isoform-specific VEGF receptor in that it only bindsVEGF₁₆₅. Like neuropilin-1, neuropilin-2 is expressed in both endotheliaand specific neurons, and is not predicted to function independently dueto its relatively short intracellular domain. The function ofneuropilin-2 in vascular development is unknown [Neufeld et al., FASEB J13:9-22 (1999); WO 99/30157].

Therapeutic Applications for VEGF Polypeptides and Antagonists

The discovery of VEGF-A as a key regulator of vascular development hasspurred active research using VEGF-based therapeutic angiogenesis incardiovascular medicine, as well as for treating diseases characterizedby pathological angiogenesis with VEGF antagonists. Subsequentidentification of additional VEGF family proteins and their roles invascularization have also led to the development of therapies based onthese growth factors [Ferrara and Alitalo, Nature Med 5:1359-1364(1999)]. Animal studies of hindlimb ischemia, and myocardial ischemiausing VEGF-A or VEGF-C, delivered by administration of recombinantprotein or gene transfer using naked DNA or adenoviral vectors,implicate these molecules in promoting vascularization and increasingcoronary blood flow. These promising results have led to clinical trialsin which patients with limb ischemia were treated by arterial orintramuscular gene transfer of naked DNA encoding VEGF165. Patients withmyocardial ischemia or Burger's disease (thromboangiitis obliterans)were also injected locally with VEGF165 plasmid DNA. Although thesetrials were not placebo-controlled, the patients showed clinicalimprovement and evidence of angiogenesis in ischemic tissues. Trialsusing gene transfer of VEGF-C naked DNA or gene therapy with VEGF121using adenoviral vectors to treat patients with myocardial ischemia arecurrently in Phase I [Ferrara, J Mol Med 77:527-543 (1999); Neufeld etal., FASEB J 13:9-22 (1999); Ferrara and Alitalo, Nature Med 5:1359-1364(1999)]. The therapeutic effects of administering recombinant VEGF-Aprotein are also being tested in ongoing clinical trials. Results from aPhase I study of patients with coronary ischemia treated withintracoronary infusion of recombinant VEGF165 show evidence of improvedperfusion and collateralization. However, in the subsequent Phase IIstudy, the patients did not show significant improvement over theplacebo-controlled group. Other potential therapeutic uses for VEGFgrowth factors include using VEGF-C to promote lymphangiogenesis inpatients whose axillary lymph nodes were removed during breast carcinomasurgery. Therapies using combinations of growth factors to promotevascularization in tissues may also prove to be preferable in treatingcertain diseases [Ferrara and Alitalo, Nature Med 5:1359-1364 (1999)].

Therapies based on inhibiting the activity of VEGF growth factors arebeing tested to treat disease states characterized by pathologicalangiogenesis. VEGF expression is upregulated in most human tumorsincluding primary breast cancer and gastric carcinoma. Studies in miceindicate that tumor-associated angiogenesis and growth of the tumorcells can be inhibited by treating the animals with monoclonalantibodies against VEGF-A. Further animal studies showed that expressionof a dominant negative VEGFR-2 mutant that prevents signaling throughthis receptor, or administration of recombinant VEGFR-1 or VEGFR-2mutants, which only contain the extracellular portion of thesereceptors, suppresses growth of several tumor cell lines. Theseencouraging results led to clinical trials using humanized high affinitymonoclonal antibodies against VEGF-A (rhuMAb VEGF) as VEGF-A inhibitors.Phase II studies using rhuMAb VEGF to treat non-small cell lungcarcinoma, colorectal carcinoma, breast, and renal cell carcinoma arecurrently ongoing. Compounds targeting inhibition of VEGF-C activity arealso being tested for therapeutic uses in cancer patients: smallmolecule inhibitors of VEGF-C are in Phase II trials, and monoclonalantibodies against VEGF-C are entering clinical trials.

Retinopathy associated with diabetes mellitus, occlusion of centralretinal vein or prematurity has been correlated with increased levels ofVEGF-A. Animal studies using monoclonal antibodies against VEGF-A orsoluble VEGFR-1 or VEGFR-2 mutants containing only the extracellulardomain fused to immunoglobulin γFc domain show suppression of retinalangiogenesis. VEGF-A is also detected in age-related maculardegeneration (AMD), and its expression is thought to be the cause ofneovascularization in this disease. Intravitreal delivery of recombinanthumanized anti-VEGF-A Fab antibody fragment or injection of2′-fluoropyrimidine RNA oligonucleotide ligands (aptamers) to treat AMDare currently in clinical trials. Compounds that inhibit the activity ofVEGF growth factors may also be used to treat other disease statesinvolving abnormal angiogenesis. These include ischemic-reperfusionrelated brain edema and injury, conditions associated with ovarianhyperplasia and hypervascularity such as the polycystic ovary syndrome,endometriosis, and ovarian hyperstimulation syndrome [Ferrara andAlitalo, Nature Med 5:1359-1364 (1999)].

From the foregoing discussion, it will be apparent that the VEGF familyof growth factors, and inhibitors thereof, have tremendous potential astherapeutics. For example, such growth factors and inhibitors are usefulto promote or inhibit angiogenesis where needed, such as in thetreatment of ischemic disorders, the promotion of wound healing, or theinhibition or elimination of neoplastic disorders that areangiogenesis-dependent. However, the various naturally-occurring membersof this growth factor family often bind multiple receptors, and thevarious known receptors are expressed on multiple cell types and haveexpression patterns that may vary depending on stage of development andthe presence or absence of pathological conditions. The biologicaleffects of any particular growth factor may be receptor-dependent,isoform dependent, and cell-type dependent. A desirable therapeuticeffect mediated through one receptor may be accompanied by undesirableside-effects mediated through another receptor. Alternatively, adesirable therapeutic effect might be enhanced through stimulation ofmultiple receptors that cannot be stimulated with any single knowngrowth factor that occurs in nature. Therefore, a need exists for novelpeptide growth factors with their own unique profile of receptor bindingand receptor-stimulating or receptor-inhibiting activities.

SUMMARY OF THE INVENTION

The present invention satisfies needs identified above by providingnovel polypeptide binding molecules for naturally occurring vascularendothelial growth factor receptors, and polynucleotides that encode thenovel polypeptides and are useful for recombinant expression of thepolypeptides. For the purpose of describing the invention, the term“vascular endothelial growth factor” and the abbreviation “VEGF”(without modifier) are used herein in a generic sense, to describe anyof a family of growth factor polypeptides including but not limited toVascular Endothelial Growth Factor-A (VEGF-A), Vascular EndothelialGrowth Factor-B (VEGF-B), Vascular Endothelial Growth Factor-C (VEGF-C),Vascular Endothelial Growth Factor-D (VEGF-D), Platelet Derived GrowthFactor-A (PDGF-A), Platelet Derived Growth Factor-B (PDGF-B), PlacentaGrowth Factor (PlGF), and virally encoded VEGF-like molecules. VEGF-A iscommonly referred to in the art as “Vascular Endothelial Growth Factor”or as “VEGF,” but for clarity shall be referred to herein as VEGF-A orreferred to as specific isoforms (e.g., VEGF₁₆₅) of VEGF-A.

For example, in one aspect, the invention provides a chimericpolypeptide comprising a plurality of peptide subunits derived from twoor more naturally-occurring vertebrate vascular endothelial growthfactor polypeptides that have different vascular endothelial growthfactor receptor binding profiles, wherein the chimeric polypeptide bindsat least one receptor of one of the naturally-occurring vascularendothelial growth factor polypeptides, and wherein the chimericpolypeptide has a different receptor binding profile than thenaturally-occurring growth factor polypeptides. Isolated and purifiedchimeric polypeptides are preferred.

In this context, the term “naturally-occurring vertebrate vascularendothelial growth factor polypeptides” means polypeptides having thefollowing characteristics:

(1) the polypeptide is encoded by genomic DNA of a vertebrate (e.g., areptile, amphibian, bird, or mammal, preferably a bird or mammal, mostpreferably a mammal; especially a primate mammal such as a monkey, ape,or human) or is encoded by the genome of a vertebrate pathogen such asmammalian pox viruses;

(2) the polypeptide comprises all or a portion of an amino acid sequencethat is expressed by a vertebrate (i.e., from transcription/translationof the vertebrate's genomic DNA or from virally-inducedtranscription/translation, in the case of polypeptides encoded by viralnucleic acids);

(3) the polypeptide or portion comprises a VEGF/PDGF homology domain(V/PHD) of about 70-150 amino acids that binds to naturally-occurringreceptors and that is characterized in part by the amino acid motif:C-X(18-28)-P-X-C-X(4)-R-C-X-G-C(1-2)-X(6-12)-C-X(30-46)-C (SEQ ID NO:1202), where X represents any amino acid and numbers in parenthesesrepresent a permissible range of amino acids (e.g., X(18-28) representsa stretch of any 18-28 amino acids; C(1-2) represents one or twocysteine residues). The V/PHD usually includes eight conserved cysteineswhich form a cysteine knot motif similar to that found in human VascularEndothelial Growth Factors A, B, C, and D (VEGF-A, -B, -C, and -D), andhuman platelet-derived growth factors (PDGFs). Preferred polypeptides orportions comprise a V/PHD that is characterized by the more particularamino acid motifC-X(22-24)-P-[PSR]-C-V-X(3)-R-C-X-G-C-C-X(6)-C-X(32-41)-C (SEQ ID NO:1203), where amino acids in brackets (e.g., [PSR]) representalternatives for a single position in the amino acid sequence; and

(4) the polypeptide binds to at least one cell surface receptor that isexpressed on endothelial cells that line vertebrate blood or lymphaticvessels or pericytes/smooth muscle cells that line and support bloodvessels. Preferred polypeptides bind to a least one cell surfacereceptor that is expressed on endothelial cells.

Thus, the term “naturally-occurring vertebrate VEGF polypeptides” meanspolypeptides that have certain specified structural and functionalproperties. The term is not intended in this context to imply a sourceof origin. Thus, recombinantly produced polypeptides that satisfy theabove criteria because they have an amino acid sequences and receptorbinding properties of VEGF polypeptides that exist in nature areconsidered “naturally-occurring”. Numerous exemplary naturally occurringvascular endothelial growth factor polypeptides are already known in theart, including but not limited to human Vascular Endothelial GrowthFactor-A (VEGF-A), Vascular Endothelial Growth Factor-B (VEGF-B),Vascular Endothelial Growth Factor-C (VEGF-C), Vascular EndothelialGrowth Factor-D (VEGF-D), Platelet Derived Growth Factor-A (PDGF-A),Platelet Derived Growth Factor-B (PDGF-B), Placenta Growth Factor(PlGF); mammalian and avian orthologs thereof (where the term “ortholog”means species homolog); and Vascular Endothelial Growth Factor E(VEGF-E), NZ2 VEGF, and the two VEGF-like proteins identified in strainsD1701 and NZ10, which have been identified in poxviruses. The use ofnaturally occurring human VEGF's is preferred for the purposes ofdeveloping chimeric molecules that are useful as human therapeutics, inorder to minimize the likelihood of developing chimerics that generatean immune response in humans. However, in many cases there is very highhomology between VEGF species orthologs, especially in receptor bindingdomains, and it is contemplated that non-human naturally occurring VEGFsalso can be used to generate chimeric molecules for use in treatinghumans. Although the invention is described herein in part withreference to particular VEGF-A/VEGF-C chimeric polypeptides, chimericpolypeptides derived from any pair, or three, or four, or more of theVEGFs described herein or their species orthologs is particularlycontemplated.

As used to describe this aspect of the invention, the term “chimericrequires that the amino acid sequence of the chimeric molecule includeat least one stretch of one or more amino acids (preferably stretches of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,or more amino acids) from each of the naturally-occurring VEGF's fromwhich it was derived. Thus, the chimeric polypeptide is a “hybrid” or“mosaic” of two or more polypeptides. By “chimeric” is meant that thepolypeptide of the invention is not identical to any naturally occurringVEGF sequence (or fragment of a natural VEGF sequence).

As used to describe this aspect of the invention, the term “derivedfrom” ” (as in “derived from two or more naturally occurring VEGFpolypeptides”) means that, when the amino acid sequences of the chimericpolypeptide and the two or more naturally occurring VEGF's are alignedusing a standard algorithm, substantially all of the amino acids in thechimeric polypeptide are aligned with an identical residue in one ormore of the naturally occurring VEGF's from which the chimeric wasderived. Standard protein alignment algorithms, for example, theclustral method [Nucl Acids Res 22:4673-80 (1994)], the Jotun-Heinmethod [Methods Enzymol 183:626-645 (1990)], or the Feng-Doolittlemethod [J Mol Evol 25:351-360 (1987)], can be used to alignnaturally-occurring vertebrate vascular endothelial growth factorpolypeptides, such alignments being greatly facilitated by the presenceof the eight highly conserved cysteines dispersed through the V/PHD.Thus, it is readily established that a chimeric polypeptide is “derivedfrom” two or more naturally occurring VEGF's by performing an alignmentusing any generally accepted protein alignment algorithm. If, afteraligning the amino acid sequences of a chimeric polypeptide and two ormore naturally occurring VEGF's using any standard algorithm,substantially all of the amino acids in the chimeric polypeptide arealigned with an identical residue in one or more of the naturallyoccurring VEGF's, then the chimeric polypeptide was derived from thenaturally occurring VEGFs. In one embodiment, all of the amino acids ofthe chimeric molecule will align in this manner. However, the use of theterm “substantially all” reflects the fact that techniques describedherein for making chimeric polypeptides will sometimes introducemutations such as insertions, deletions, or substitutions, preventing100% correlation to parent sequences. In such cases, at least about 90%,92%, 94%, 95%. 96%, 97%, 98%, or 99% of the residues of the chimericpolypeptide will align with identical residues from at least one of thenatural VEGF's.

When presented with a chimeric polypeptide of the invention that alignsperfectly or substantially with the natural VEGF polypeptides from whichit was derived, it is within the skill of the art to intentionallyintroduce mutations (especially conserved mutations) into the chimericpolypeptide and test such a modified chimeric polypeptide for itsreceptor binding profile. Modifications of chimeric polypeptides(especially conserved amino acid substitutions) that do not introducesubstantial changes in receptor binding profile are intended asequivalents within the scope of the present invention.

In the context of such chimeric polypeptides, the term “plurality ofpeptide subunits” means two or more peptide subunits. Exemplified hereinare chimeric polypeptides obtained by fragmenting two naturallyoccurring VEGF cDNA's (human VEGF-A and human VEGF-C) into nine subunitsof about 8-16 codons each, recombining these fragments into all 512permutations of the nine subunits (maintaining subunit order), andexpressing the resultant chimeric cDNAs. The number and the size offragments is not intended as a critical feature. In preferredembodiments, plurality comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more subunits. Asexemplified herein, the “subunits” are joined by peptide bonds to form apolypeptide chain.

In the context of chimeric polypeptides of the invention or naturallyoccurring VEGF polypeptides, determination of “vascular endothelialgrowth factor receptor binding profile” means the determination of thereceptors to which a polypeptide will bind and the receptors to which itwill not. Known VEGF receptors, including VEGFR-1, VEGFR-2, and VEGFR-3,are described in greater detail elsewhere herein. Known PDGF receptorsare also described in greater detail elsewhere herein. Where a chimericpolypeptide has been derived in part from a naturally occurring PDGFsequence, screening the chimeric polypeptide for binding to PDGFreceptors is contemplated as part of the receptor binding profiledetermination.) By way of example, if a chimeric polypeptide was derivedfrom a VEGF-A that binds to VEGFR-1 and VEGFR-2 and from a VEGF-C thatbinds to VEGFR-2 and VEGFR-3, the chimeric polypeptide has a differentreceptor binding profile than either of its parent molecules if it bindsto only one of the three receptors, or if it binds to all threereceptors, or if it binds to VEGFR-1 and VEGFR-3 but not VEGFR-2. In onepreferred embodiment, the invention provides chimeric polypeptideswherein the chimeric polypeptide binds to at least two VEGF receptorsbound by the two or more naturally occurring vertebrate VEGFpolypeptides, and wherein each of the naturally-occurring VEGFpolypeptides from which the chimeric polypeptide was derived fail tobind to one or more of the at least two VEGF polypeptides.

Screening polypeptides of the invention for binding to the neuropilinsNP-1 and NP-2 are not contemplated as part of the receptor bindingprofile determination, because the portions of VEGF (and other familymembers) responsible for NP-1 and NP-2 binding are portions outside ofthe V/PHD core region. NP-1 binding is mediated by amino acid residues142 to 185 of SEQ ID NO: 2 for VEGF-A, and amino acid residues 138 to182 for VEGF-B [Soker et al., J Biol Chem 271:5761-7 (1996); Makinen etal., J Biol Chem 274:21217-22 (1999)]. As explained below, addition ofupstream or downstream sequences to chimeric polypeptides of theinvention is contemplated, and some added sequences are contemplated toresult in NP-1 or NP-2 binding.

The present invention is believed to provide the first disclosure of apolypeptide that is capable of binding to all of VEGFR-1, VEGFR-2, andVEGFR-3. All polypeptides having this receptor binding profile areintended as within the scope of the invention.

Naturally occurring VEGF polypeptides generally bind their respectivereceptors with high affinity, which is generally understood in thiscontext to mean binding with a sub-nanomolar dissociation constant. Forexample, VEGF-A binds VEGFR-1 and VEGFR-2 with Kd of approximately 16 pMand 760 pM, respectively; and VEGF-C binds VEGFR-2 and VEGFR-3 with Kdof approximately 410 pM and 135 pM, respectively. Because it is possibleto administer a therapeutic growth factor protein to achieveconcentrations exceeding normal serum concentrations, and to formulatesuch polypeptides to increase biological half-life, it is contemplatedthat chimeric polypeptides having less receptor affinity (i.e., higherdissociation constants) nonetheless will be useful as receptor agonistsand antagonists. For the purposes of scoring receptor binding ofchimeric polypeptides, a 50 nanomolar dissociation constant cutoff isselected. Chimeric polypeptides that bind a receptor with a dissociationconstant of less than 50 nanomolar as determined by any conventional andrecognized method, such as those described in Coligan et al., CurrentProtocols in Protein Science, Vol. 2, New York, John Wiley & Sons, Inc.,p. A.5A.1-A.5A.40 (1998), incorporated herein by reference, is scored asbinding to a receptor, and polypeptides with lower affinities are scoredas non-binding.

It is well known in the literature that naturally occurring VEGF's areexpressed as splice variants and/or as pre-protein molecules and/or asprepro-protein molecules that undergo proteolytic processing. Chimericpolypeptides of the invention include chimeric (hybrid) receptor bindingdomains as explained in the preceding paragraphs, and optionally mayinclude additional upstream or downstream sequences from naturallyoccurring VEGF's, including upstream and downstream sequences that arepresent in mature isoforms of naturally occurring circulating VEGF's;and/or upstream or downstream pro-peptide sequences that are removedduring normal intracellular or extracellular processing. By way ofillustration, the chimeric polypeptides described in Example 1 wereprepared using residues 34-135 (SEQ ID NO: 2) of VEGF-A and using112-216 of human prepro-VEGF-C (SEQ ID NO: 22). Chimeric polypeptides ofthe invention include the peptides actually exemplified, and alsoinclude such peptides modified by the addition of upstream or downstreamVEGF-A or VEGF-C sequences from SEQ ID NOs: 2 or 22. With respect toVEGF-A/VEGF-C chimeric polypeptides as exemplified herein, the additionof upstream and downstream sequences that correspond with amino- and/orcarboxyl-terminal sequences characteristic of natural VEGF-A or VEGF-Cisoforms is particularly contemplated.

It is also well known in the literature to recombinantly expressproteins with an initiator methionine, with a heterologous signalpeptide, with one or more tag sequences to facilitate purification, asfusions with other polypeptides, and the like. It is also well known tomodify polypeptides with glycosylation, pegylation, or othermodifications, some of which improve stability, circulating half-life,or (in the case of glycosylation) may make the polypeptide more similarto endogenous vascular endothelial growth factors. Chimeric polypeptidesaccording to the invention may comprise any such modifications andadditions to the amino acid sequence derived from two or morenaturally-occurring vertebrate vascular endothelial growth factorpolypeptides.

In addition to chimeric molecules having different receptor bindingprofiles, an additional aspect of the invention includes chimericmolecules having increased receptor binding affinity. For example, theinvention provides a chimeric polypeptide comprising a plurality ofpeptide subunits derived from two or more naturally-occurring vertebratevascular endothelial growth factor polypeptides, wherein the chimericpolypeptide binds at least one naturally-occurring vascular endothelialgrowth factor receptor with an increased binding affinity compared tothe binding affinity of the two or more naturally-occurring vascularendothelial growth factors for the receptor. Chimeric molecules thatbind a receptor with greater affinity than naturally occurring VEGF'sare among the preferred chimeric molecules of the invention, even if thereceptor binding profile for the chimeric molecules is identical to thatof a naturally occurring VEGF. Increased receptor binding affinity isexpected to correlate with great potency as receptor activators orinhibitors. Generally, dissociation constants (K_(d)) determined by anyaccepted procedure are indicative of receptor affinity, with lower K_(d)indicative of greater binding affinity. Particularly contemplated arechimeric molecules that display any reduction in K_(d) that isstatistically significant at a level of p<0.05 in side-by-side tests[see, e.g., Coligan et al., Current Protocols in Protein Science, Vol.2, New York, John Wiley & Sons, Inc., p. A.5A.1-A.5A.40 (1998)] comparedto naturally-occurring molecules from which the chimera was derived.Chimeras that show a twenty percent reduction in Kd (i.e., increasedbinding affinity) with respect to a VEGF receptor are preferred.Reductions of 33% or 50% are highly preferred. A 3-fold reduction (e.g.,a Kd of 33.3 pM for a chimeric polypeptide compared to a 100 pM Kd of anaturally occurring VEGF), 5-fold reduction, 10-fold reduction, or20-fold reduction in dissociation constants or is very highly preferred.

Another related preferred class of chimeric molecules are thosemolecules that display any reduction in EC₅₀ concentration that isstatistically significant at a level of p<0.05 in side-by-side testscompared to naturally-occurring molecules from which the chimera wasderived. Chimeras that show a twenty percent reduction in EC₅₀ (i.e.,increased binding affinity) with respect to a VEGF receptor arepreferred. Reductions of 33% or 50% are highly preferred. EC₅₀, or thehalf effective concentration, is the concentration that produces 50% ofa maximal effect. An exemplary assay for determining the EC₅₀ of aputative ligand for a specific receptor is set forth in Example 6,below.

The Examples set forth below provide a description of the synthesis andassaying of numerous specific hybrid polypeptides of the invention,every one of which is itself intended as an aspect of the invention. Apreferred group of hybrid polypeptides from among the exemplifiedpolypeptides are polypeptides that comprise an amino acid sequence ofthe formula:NH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH

wherein X₁ comprises an amino acid sequence selected from the groupconsisting of amino acids 3-11 of SEQ ID NO: 128 and amino acids 3-11 ofSEQ ID NO:137; wherein X₂ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 129 and 138; wherein X₃ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:130 and 139; wherein X₄ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 131 and 140; wherein X₅ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:132 and 141; wherein X₆ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 133 and 142; wherein X₇ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:134 and 143; wherein X₈ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 135 and 144; wherein X₉ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:136 and 145; wherein NH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH is notidentical to amino acids 34 to 135 of SEQ ID NO: 2 or amino acids 112 to216 of SEQ ID NO: 22; and wherein the polypeptide binds to at least onereceptor selected from the group consisting of human VEGFR-1, humanVEGFR-2, and human VEGFR-3. As described below in greater detail, eachof the specified amino acid sequence pairs (e.g., SEQ ID NO: 128 and137) are encoded by a VEGF-A cDNA fragment or a VEGF-C cDNA fragmentthat align with each other when the receptor binding domains of VEGF-Aand VEGF-C are aligned to maximize sequence homology, using standardalignment algorithms.

With respect to the foregoing genus of hybrid of polypeptides, onepreferred subgenus comprises those polypeptides which bind to exactlyone receptor selected from the group consisting of human VEGFR-1, humanVEGFR-2, and human VEGFR-3. Initial screens suggest that the followingspecific constructs (described below in detail) satisfy this criteria:82-14, 82-16, 22-3, 72-6, 12-14, 12-16, 32-9, 32-11, 32-14, 32-15,32-16, 52-9, 52-11, 52-14, 52-15, 14-7, 23-10, 23-12, 23-14, 33-1, 33-3,33-6, 33-9, 53-1, 53-3, 53-7, 62-8, 62-10, 62-13, 63-3, 63-6, 73-7,73-15, -8, 74-10, 74-12, 11-9, 11-13, 12-1, 12-5, 81-9, 81-13, 13-9,13-11, 13-13, 13-15, 14-1, 14-5, 41-1, 43-1, 83-9, 83-13, 83-15, 61-1,61-3, 62-1, 82-5, 84-1, 84-5.

Another preferred subgenus comprises those hybrid polypeptides that bindto VEGFR-1 and VEGFR-3, but not to VEGFR-2. Initial screens suggest thatthe following specific constructs satisfy this criteria: 12-9, 12-13,14-9, 82-9, 82-13, 84-9.

A highly preferred subgenus comprises hybrid polypeptides of theinvention that bind VEGFR-1, VEGFR-2, and VEGFR-3. Initial screenssuggest that the following specific constructs satisfy this criteria:12-7, 12-11, 82-11, 84-11.

Other subgenuses include hybrid polypeptides that bind VEGFR-1 andVEGFR-2 (both of which are bound by VEGF-A) but not VEGFR-3; and hybridpolypeptides that bind VEGFR-2 and VEGFR-3 (both of which are bound byfully processed VEGF-C) but not VEGFR-1.

As taught in greater detail below, the fourth fragment (X₄) appears toinclude residues that are important for conferring VEGFR-3 bindingaffinity. For this reason, another preferred genus of the hybridpolypeptides are those wherein X₄ comprises SEQ ID NO: 140, and whereinthe polypeptide binds to VEGFR-3. Fragments 5 and 8 of VEGF-C alsoappear to contribute to VEGFR-3 binding. Thus, highly preferred arepolypeptides further characterized by X₅ comprising SEQ ID NO: 141,and/or X₈ comprising SEQ ID NO: 144.

Similarly, the data below suggests that fragments 2 and 7 of VEGF-Acontribute to VEGF-R1 binding. Thus, another preferred gene of thehybrid polypeptides are those wherein X₂ comprises SEQ ID NO: 129, andwherein the polypeptide binds to VEGFR-1. In a highly preferredembodiment, X₇ comprises SEQ ID NO: 134. In an embodiment where it isdesirous for this polypeptide also to bind to VEGFR-3, a preferredconstruct is one wherein X₄ comprises SEQ ID NO: 140. To confer VEGFR-3binding, it is still more preferable for X₅ to comprise SEQ ID NO: 141,and/or for X₈ to comprise SEQ ID NO: 144.

The recombination experiments described below to generate hybridmolecules were performed only with receptor binding domains of humanVEGF-A and VEGF-C, rather than with sequences corresponding to naturalsecreted forms of VEGF-A and VEGF-C or pre-protein or prepro-proteinsequences. However, routine recombinant DNA techniques, such as thosedescribed in Ausubel, et al. (Eds.), Protocols in Molecular Biology,John Wiley & Sons (1994-1999) or Sambrook et al., (Eds.), MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y. (1989), can be used to join polynucleotides encodingthe hybrid proteins with polynucleotides encoding VEGF-A or VEGF-Csequences that are found upstream or downstream of the receptor bindingdomain in naturally-occurring proteins, especially sequences found innaturally-occurring secreted and circulating forms of VEGF-A or VEGF-C.

Thus, the invention provides a polypeptide comprising an amino acidsequence of the formula X_(N)-V/PHD-X_(C), wherein X_(N) is selectedfrom the group consisting of amino acids 1-34 of SEQ ID NO: 2, aminoacids 1-111 of SEQ ID NO: 22, amino acids 1-34 of SEQ ID NO: 147, orfragments thereof;

wherein V/PHD is a chimeric polypeptide as described elsewhere herein;

wherein X_(C) is selected from the group consisting of amino acids136-191 of SEQ ID NO: 2, amino acids 217-419 of SEQ ID NO: 22, aminoacids 136-232 of SEQ ID NO: 147, or fragments thereof; and

wherein X_(N) and X_(C) are each identical to amino acid sequence in anaturally occurring human VEGF-A or VEGF-C precursor protein or anaturally occurring human VEGF-A or VEGF-C isoform.

In one specific variation, the invention provides hybrid polypeptides asdescribed above, wherein the polypeptide further includes one or moreamino acid sequences selected from the group consisting of aprepro-VEGF-C signal peptide, a prepro-VEGF-C amino-terminal propeptide,and a prepro-VEGF-C carboxy-terminal pro-peptide.

Expression of hybrid polypeptides of the invention is not restricted toexpression only with naturally-occurring flanking VEGF-A or VEGF-Csequences, however. Expression of polypeptides of the invention inbacteria may be accomplished by including an initiator methionine ormethionine-lysine upstream of the hybrid VEGF sequences, whereasexpression and secretion in mammalian cells is most convenientlyaccomplished by including at least a signal peptide. Thus, in oneembodiment, the invention provides a polypeptide as described above,wherein the polypeptide further includes an amino terminal methionineresidue or an amino-terminal Met-Lys sequence. In another embodiment,the polypeptide further includes a signal peptide amino acid sequenceconnected to the amino acid sequence of the formulaNH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH.

Expression of polypeptides of the invention as fusions with otherheterologous sequences, such as tag sequences to facilitate purificationor expression as part of larger fusion peptides also is contemplated. Anexemplary tag of this type is a poly-histidine sequence, generallyaround six histidine residues, that permits isolation of a compound solabeled using nickel chelation. Other labels and tags, such as the FLAG®tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used inthe art, are embraced by the invention. Exemplary fusions include use ofcommercially available vectors that express a desired polypeptide aspart of glutathione-5-transferase (GST) fusion product. After cleavageof the GST component from the desired polypeptide, an additional glycineresidue at position −1 may remain. Variants which result from expressionin other vector systems are also contemplated.

By virtue of the receptor binding and activity assays described herein,the present application also provides variants (analogs) of the hybridpolypeptides of the invention, wherein one or more amino acids of thehybrid peptide amino acid sequence has been added, deleted, orsubstituted by another amino acid, and wherein the hybrid retains thereceptor binding and/or a biological activity characteristic of thehybrid polypeptide.

Substitution variants wherein merely conservative substitutions havebeen introduced (e.g., by modification of polynucleotides encodingpolypeptides of the invention) are intended as equivalents of hybridpolypeptides of the invention. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions based on aminoacid side chain properties are set out in the table immediately below,using standard one letter abbreviations. SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G, A, P, I, L, V Polar-uncharged C, S, T, M, N,Q Polar-charged D, E, K, R Aromatic F, W, Y Other N, Q, D, E

Alternatively, conservative amino acids can be grouped as described inLehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY(1975), pp. 71-77] as set out in the table immediately below. Non-polar(hydrophobic) side chain Aliphatic: A, L, I, V, P Aromatic: F, WSulfur-containing: M Borderline: G Uncharged-polar side chain Hydroxyl:S, T, Y Amides: N, Q Sulfhydryl: C Borderline: G Positively Charged(Basic): K, R, H Negatively Charged (Acidic): D, E

The following table provides still an another alternative, exemplary setof conservative amino acid substitutions. Both one letter and threeletter abbreviations are shown: Original Residue ConservativeSubstitutions Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

For many proteins, the effects of any individual or small group of aminoacid changes is unlikely to significantly alter biological properties,especially if the changes are conservative substitutions, provided thechanges are not introduced at critical residues. Preferred variants ofthe hybrid polypeptides of the invention share at least about 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with hybrids thatconsist entirely of amino acid sequences derived from naturallyoccurring VEGF's.

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J.Applied Math., 48: 1073 (1988).

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux et al.,Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,J. Mol. Biol., 215:403-410 (1990)). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., supra). The well known Smith Watermanalgorithm may also be used to determine identity.

Preferred parameters for a polypeptide sequence comparison include thefollowing:

-   -   Algorithm: Needleman et al., J. Mol. Biol., 48, 443-453 (1970);    -   Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl.        Acad. Sci. USA, 89: 10915-10919 (1992);    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0

Preferred parameters for nucleic acid molecule sequence comparisonsinclude the following:

Algorithm: Needleman et al., J. Mol. Biol., 48: 443-453 (1970);

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Thus, in still another embodiment, the invention provides a polypeptidecomprising a non-naturally occurring vascular endothelial growth factoramino acid sequence, wherein said non-naturally occurring vascularendothelial growth factor amino acid sequence consists of an amino acidsequence that is at least 95% identical to an amino acid sequence of theformula:NH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH

wherein X₁ comprises an amino acid sequence selected from the groupconsisting of amino acids 3-11 of SEQ ID NO: 128 and amino acids 3-11 ofSEQ ID NO: 137; wherein X₂ comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 129 and 138; wherein X₃comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 130 and 139; wherein X₄ comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 131 and 140; whereinX₅ comprises an amino acid sequence selected from the group consistingof SEQ ID NOs: 132 and 141; wherein X₆ comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 133 and 142; whereinX₇ comprises an amino acid sequence selected from the group consistingof SEQ ID NOs: 134 and 143; wherein X₈ comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 135 and 144; whereinX₉ comprises an amino acid sequence selected from the group consistingof SEQ ID NOs: 136 and 145; and wherein the polypeptide binds to atleast one receptor selected from the group consisting of human VEGFR-1,human VEGFR-2, and human VEGFR-3. In a preferred embodiment,NH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH is not identical to amino acids 34to 135 of SEQ ID NO: 2 or amino acids 112 to 216 of SEQ ID NO: 22.

By “non-naturally occurring vascular endothelial growth factor aminoacid sequence” is meant a sequence that is not identical to any known,naturally occurring amino acid sequence, such as, in this case, receptorbinding domains from known VEGF-A or VEGF-C sequences.

Stated more generally, the invention provides a polypeptide thatcomprises an amino acid sequence that is at least about 70%, 80%, 90%,95%, 96%, 97%, 98%, or 99% identical to any specific amino acid sequenceof the invention, and that binds at least one of the naturally-occurringvascular endothelial growth factor or platelet derived growth factorreceptors, and that has a different receptor binding profile or animproved receptor binding affinity than a naturally-occurring growthfactor polypeptide. Polypeptides that satisfy the percent identitycriteria and that display the same receptor binding profile as thereferent polypeptide are especially contemplated. For example, theinvention provides a polypeptide that comprises an amino acid sequencethat is at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequences encoded by constructs 12-7 (SEQ IDNO: 63), 12-11 (SEQ ID NO: 71), 82-11, or 84-11, wherein the polypeptidebinds VEGFR-1, VEGFR-2, and VEGFR-3.

In yet another aspect, the invention provides a dimeric protein moleculecomprising a first polypeptide associated with a second polypeptide,wherein at least one of the polypeptides is a polypeptide according tothe present invention. The association between the polypeptides may beby way of covalent bonding (e.g., disulfide bonding) or non-covalentbonding of polypeptide chains (e.g, hydrogen bonding, bonding due tostable or induced dipole-dipole interactions, bonding due to hydrophobicor hydrophilic interactions, combinations of these bonding mechanisms,and the like).

In another embodiment, the invention provides polynucleotides (e.g.,cDNA, cDNA with introns introduced to facilitate expression ineukaryotic systems, synthetic DNA, RNA, or combinations thereof, singleor double stranded) that comprise a nucleotide sequence encoding theamino acid sequence of the polypeptides of the invention. Purified andisolated polynucleotides are preferred. Due to the well-known degeneracyof the genetic code, several polynucleotides sequences exist that encodeeach polypeptide amino acid sequence of the invention. Suchpolynucleotides are useful for recombinantly expressing the polypeptidesof the invention.

The invention also embraces polynucleotides that encode VEGF receptorbinding polypeptides and that hybridize under moderately stringent orhigh stringency conditions to the complete non-coding strand, orcomplement, of the polynucleotides specifically described herein thatencode VEGF receptor binding polypeptides. This genus of polynucleotidesembraces polynucleotides that encode polypeptides with one or a fewamino acid differences (additions, insertions, or deletions) relative toamino acid sequences specifically taught herein. Such changes are easilyintroduced by performing site directed mutagenesis, for example, or bysubstituting a fragment from a non-human ortholog VEGF-A or VEGF-Cpolypeptide for a fragment of a human VEGF-A or VEGF-C polypeptide usedto construct the hybrid polypeptides of the invention.

Exemplary highly stringent hybridization conditions are as follows:hybridization at 65° C. for at least 12 hours in a hybridizationsolution comprising 5×SSPE, 5×Denhardt's, 0.5% SDS, and 2 mg sonicatednon-homologous DNA per 100 ml of hybridization solution; washing twicefor 10 minutes at room temperature in a wash solution comprising 2×SSPEand 0.1% SDS; followed by washing once for 15 minutes at 65° C. with2×SSPE and 0.1% SDS; followed by a final wash for 10 minutes at 65° C.with 0.1×SSPE and 0.1% SDS. Moderate stringency washes can be achievedby washing with 0.5×SSPE instead of 0.1×SSPE in the final 10 minute washat 65° C. Low stringency washes can be achieved by using 1×SSPE for the15 minute wash at 65 C, and omitting the final 10 minute wash. It isunderstood in the art that conditions of equivalent stringency can beachieved through variation of temperature and buffer, or saltconcentration as described Ausubel, et al. (Eds.), Protocols inMolecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.Modifications in hybridization conditions can be empirically determinedor precisely calculated based on the length and the percentage ofguanosine/cytosine (GC) base pairing of the probe. The hybridizationconditions can be calculated as described in Sambrook et al., (Eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51. For example,the invention provides a polynucleotide that comprises a nucleotidesequence that hybridizes under moderately stringent or high stringencyhybridization conditions to any specific nucleotide sequence of theinvention, and that encodes a polypeptide that binds at least one of thenaturally-occurring vascular endothelial growth factor or plateletderived growth factor receptors, and that has a different receptorbinding profile or an improved receptor binding affinity than anaturally-occurring growth factor polypeptide. Polynucleotides thatsatisfy the hybridization criteria and that display the same receptorbinding profile as the referent polynucleotide are especiallycontemplated. For example, the invention provides a polynucleotide thatcomprises a nucleotide sequence that hybridizes under moderatelystringent or high stringency hybridization conditions to the nucleotidesequences taught herein for constructs 12-7 (SEQ ID NO: 62), 12-11 (SEQID NO: 70), 82-11, or 84-11, wherein the polynucleotide encodes apolypeptide that binds VEGFR-1, VEGFR-2, and VEGFR-3.

In a related embodiment, the invention provides a polynucleotide thatcomprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, or 99% identical to any specific nucleotide sequence of theinvention, and that encodes a polypeptide that binds at least one of thenaturally-occurring vascular endothelial growth factor or plateletderived growth factor receptors, and that has a different receptorbinding profile or an improved receptor binding affinity than anaturally-occurring growth factor polypeptide. Polynucleotides thatsatisfy the percent identity criteria and that display the same receptorbinding profile as the referent polynucleotide are especiallycontemplated. For example, the invention provides a polynucleotide thatcomprises a nucleotide sequence that is at least 95% identical to thenucleotide sequences taught herein for constructs 12-7 (SEQ ID NO: 62),12-11 (SEQ ID NO: 70), 82-11, or 84-11, wherein the polynucleotideencodes a polypeptide that binds VEGFR-1, VEGFR-2, and VEGFR-3.

In a related embodiment, the invention provides vectors comprising apolynucleotide of the invention. Such vectors are useful, e.g., foramplifying the polynucleotides in host cells to create useful quantitiesthereof. In preferred embodiments, the vector is an expression vectorwherein the polynucleotide of the invention is operatively linked to apolynucleotide comprising an expression control sequence. Autonomouslyreplicating recombinant expression constructs such as plasmid and viralDNA vectors incorporating polynucleotides of the invention arespecifically contemplated. Expression control DNA sequences includepromoters, enhancers, and operators, and are generally selected based onthe expression systems in which the expression construct is to beutilized. Preferred promoter and enhancer sequences are generallyselected for the ability to increase gene expression, while operatorsequences are generally selected for the ability to regulate geneexpression. Expression vectors are useful for recombinant production ofpolypeptides of the invention. Expression constructs of the inventionmay also include sequences encoding one or more selectable markers thatpermit identification of host cells bearing the construct. Expressionconstructs may also include sequences that facilitate, and preferablypromote, homologous recombination in a host cell. Preferred constructsof the invention also include sequences necessary for replication in ahost cell.

Vectors also are useful for “gene therapy” treatment regimens, wherein apolynucleotide that encodes a polypeptide of the invention is introducedinto a subject in need of treatment involving the modulation(stimulation or blockage) of vascular endothelial growth factorreceptors, in a form that causes cells in the subject to express thepolypeptide of the invention in vivo.

In another related embodiment, the invention provides host cells,including prokaryotic and eukaryotic cells, that are transformed ortransfected (stably or transiently) with polynucleotides of theinvention or vectors of the invention. Polynucleotides of the inventionmay be introduced into the host cell as part of a circular plasmid, oras linear DNA comprising an isolated protein coding region or a viralvector. Methods for introducing DNA into the host cell well known androutinely practiced in the art include transformation, transfection,electroporation, nuclear injection, or fusion with carriers such asliposomes, micelles, ghost cells, and protoplasts. As stated above, suchhost cells are useful for amplifying the polynucleotides and also forexpressing the polypeptides of the invention encoded by thepolynucleotide. Such host cells are useful in assays as describedherein. For expression of polypeptides of the invention, any host cellis acceptable, including but not limited to bacterial, yeast, plant,invertebrate (e.g., insect), vertebrate, and mammalian host cells. Fordeveloping therapeutic preparations, expression in mammalian cell lines,especially human cell lines, is preferred. Use of mammalian host cellsis expected to provide for such post-translational modifications (e.g.,glycosylation, truncation, lipidation, and phosphorylation) as may bedesirable to confer optimal biological activity on recombinantexpression products of the invention. Glycosylated and non-glycosylatedforms of polypeptides are embraced by the present invention. Similarly,the invention further embraces polypeptides described above that havebeen covalently modified to include one or more water soluble polymerattachments such as polyethylene glycol, polyoxyethylene glycol, orpolypropylene glycol.

Polypeptides of the invention also may be chemically synthesized.

In still another related embodiment, the invention provides a method forproducing a vascular endothelial growth factor receptor binding protein,comprising the steps of growing a host cell of the invention in anutrient medium and isolating the polypeptide or variant thereof fromthe cell or the medium. Isolation of the polypeptide from the cells orfrom the medium in which the cells are grown is accomplished bypurification methods known in the art, e.g., conventionalchromatographic methods including immunoaffinity chromatography,receptor affinity chromatography, hydrophobic interactionchromatography, lectin affinity chromatography, size exclusionfiltration, cation or anion exchange chromatography, high pressureliquid chromatography (HPLC), reverse phase HPLC, and the like. Stillother methods of purification include those wherein the desired proteinis expressed and purified as a fusion protein having a specific tag,label, or chelating moiety that is recognized by a specific bindingpartner or agent. The purified protein can be cleaved to yield thedesired protein, or be left as an intact fusion protein. Cleavage of thefusion component may produce a form of the desired protein havingadditional amino acid residues as a result of the cleavage process.

Also within the scope of the invention are compositions comprisingpolypeptides or polynucleotides of the invention. In a preferredembodiment, such compositions comprise one or more polynucleotides orpolypeptides of the invention that have been formulated with apharmaceutically acceptable (e.g., sterile and non-toxic) diluent orcarrier. Liquid, semisolid, or solid diluents that serve aspharmaceutical vehicles, excipients, or media are preferred. Any diluentknown in the art may be used. Exemplary diluents include, but are notlimited to, water, saline solutions, polyoxyethylene sorbitanmonolaurate, magnesium stearate, methyl- and propylhydroxybenzoate,talc, alginates, starches, lactose, sucrose, dextrose, sorbitol,mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.Such formulations are useful, e.g., for administration of polypeptidesor polynucleotides of the invention to mammalian (including human)subjects in therapeutic regimens.

Similarly, the invention provides for the use of polypeptides orpolynucleotides of the invention in the manufacture of a medicament forthe treatment of disorders described herein, including but not limitedto disorders characterized by undesirable endothelial cell proliferationand/or disorders characterized by ischemia and/or vessel occlusion,wherein neovascularization is desirable.

In a related embodiment, the invention provides a kit comprising apolynucleotide, polypeptide, or composition of the invention packaged ina container, such as a vial or bottle, and further comprising a labelattached to or packaged with the container, the label describing thecontents of the container and providing indications and/or instructionsregarding use of the contents of the container to treat one or moredisease states as described herein.

In yet another aspect, the present invention provides methods ofproducing polypeptides having novel VEGF receptor binding andstimulation properties, and methods for producing polynucleotides thatencodes such polypeptides. For example, the invention provides a methodfor making a polynucleotide that encodes a polypeptide that modulatesthe growth of mammalian endothelial cells or mammalian pericytes/smoothmuscle cells; comprising the steps of: preparing polynucleotides thatencode amino acid fragments of at least two vertebrate vascularendothelial growth factor polypeptides; commingling the polynucleotidesunder conditions wherein the polynucleotides recombine to form hybridpolynucleotides; expressing the hybrid polynucleotides to make hybridpolypeptides encoded by the hybrid polynucleotides; screening the hybridpolypeptides to identify a hybrid polypeptide that binds to a receptorfor a vertebrate vascular endothelial growth factor; and selecting thepolynucleotide that encodes the hybrid polypeptide that binds to thereceptor in the screening step. Expression of the selectedpolynucleotide (to produce the desired polypeptide) also iscontemplated.

In this context, “modulate the growth of mammalian endothelial cells”means stimulate such growth by inducing a mitogenic signal throughbinding cell surface receptors expressed on vascular endothelial cells,or inhibiting such growth. As explained elsewhere herein, inhibition maybe due to blockage of vascular endothelial growth factor receptors, orthe formation of heterodimers with endogenous growth factors thatprevent stimulation of endogenous receptors by the endogenous growthfactors. Inhibition also may be achieved by conjugating cytotoxic agentsto polypeptides of the invention that bind VEGF receptors. Exemplarytoxins are known in the art and described elsewhere herein. Polypeptidesof the invention conjugated to cytotoxic agents or other agents thatmodulate cell growth are contemplated as another aspect of theinvention.

In this context, “vertebrate vascular endothelial growth factorpolypeptides” again means polypeptides having the followingcharacteristics:

(1) the polypeptide is encoded by genomic DNA of a vertebrate (e.g., areptile, amphibian, bird, or mammal, preferably a bird or mammal, mostpreferably a mammal; especially a primate mammal such as a monkey, ape,or human) or is encoded by the genome of a vertebrate pathogen such asmammalian pox viruses;

(2) the polypeptide comprises all or a portion of a coding sequence thatis expressed by a vertebrate (i.e., from transcription/translation ofthe vertebrate's genomic DNA or from virally-inducedtranscription/translation, in the case of polypeptides encoded by viralnucleic acids);

(3) the polypeptide or portion comprises a VEGF homology domain (V/PHD)of about 70-150 amino acids that binds to naturally occurring receptorsand that is characterized in part by the amino acid motif:C-X(18-28)-P-X-C-X(4)-R-C-X-G-C(1-2)-X(6-12)-C-X(30-46)-C, where Xrepresents any amino acid and numbers in parentheses represent apermissible range of amino acids (e.g., X(18-28) represents a stretch ofany 18-28 amino acids; C(1-2) represents one or two cysteine residues).The V/PHD includes eight conserved cysteines which form a cysteine knotmotif similar to that found in human vascular endothelial growth factorsA, B, C, and D (VEGF-A, -B, -C, and -D, and human platelet-derivedgrowth factor (PDGF). Preferred polypeptides or portions comprise a VPHDthat is characterized by the more particular amino acid motifC-X(22-24)-P-[PSR]-C-V-X(3)-R-C-X-G-C-C-X(6)-C-X(32-41)-C, where aminoacids in brackets (e.g., [PSR]) represent alternatives for a singleposition in the amino acid sequence; and

(4) the polypeptide binds to at least one cell surface receptor that isexpressed on endothelial cells that line vertebrate blood or lymphaticvessels or pericytes/smooth muscle cells that line and support bloodvessels. Preferred polypeptides bind to a least one cell surfacereceptor that is expressed on endothelial cells.

Several methods exist for practicing the preparing step. In onevariation, single-stranded oligonucleotides are prepared based onknowledge of mammalian VEGF polypeptide sequences and the universalgenetic code and using conventional chemical synthesis techniques.Example 1 below demonstrates such a technique, wherein syntheticoligonucleotide pairs were prepared and annealed to preparedouble-stranded polynucleotides having single-stranded cohesive endsthat encoded fragments of human VEGF-A and human VEGF-C. In anothervariation, cDNAs or genomic DNAs (preferably cDNAs) encoding naturalVEGF's are fragmented using one or more restriction endonucleases, usingDNaseI, or using Exonuclease III. [See, e.g., Chang et al., NatureBiotechnology, 17: 793-797 (1999) (DNaseI procedure); Kikuchi et al.,Gene, 236: 159-167 (1999) (restriction endonuclease procedure); Harayamaet al., TIBTECH, 16: 76-82 (1998) (review); Patten et al., Curr. Opin.Biotechnology, 8: 724-733 (1997) (review, DNase I); Zhang et al., Proc.Natl. Acad. Sci. USA, 94: 4504-09 (1997) (DNase I procedure); Stemmer,Proc. Natl. Accd. Sci. USA, 91: 10747-1074 (1997) (DNase I procedure);Stemmer, Nature, 370: 389-391 (1994) (DNase I procedure); and Ostermeieret al., Nature Biotechnology, 17: 1205-1209 (1999) (ExoIII procedure),all incorporated herein by reference in their entirety]. In stillanother variation, a cDNA (coding or non-coding strand) is used as atemplate to synthesize complementary fragments, using DNA polymerase andchain-termination reagents. [See, e.g., Lehtovaara et al., ProteinEngineering, 2: 63-68 (1988), incorporated by reference.]

Several methods also exist for practicing the commingling step. In onevariation, the polynucleotides are prepared with complementary cohesivesingle-stranded ends, to facilitate annealing of fragments in a desiredorder under conventional annealing and ligation conditions forpolynucleotides. Example 1 below provides a demonstration of thistechnique to generate 510 human VEGF-A/VEGF-C hybrids. Such a techniquealso may be suitable for annealing fragment mixtures of two or more VEGFcDNAs that have been digested with restriction endonucleases.Alternatively, the commingling step is accomplished by mixing thepolynucleotides and subjecting them to a self-priming PCR reaction thatinvolves successive steps of denaturation, annealing, and extension.[See, e.g., Chang et al. (1999); Kikuchi et al. (1999); Patten et al.(1997); Zhang et al. (1997); Stemmer Proc. Natl. Accd. Sci. USA, 91:10747-1074 (1994); and Stemmer, Nature, 370: 389-391 (1994).].Optionally, the PCR is performed under conditions that introduce errors(mutations) in the PCR products. Such mutations introduce additionalmolecular variation, and are expected to reduce the overall percentageof biologically active molecules, but also may produce molecules withunexpectedly superior activities.

After synthesizing the hybrid DNA molecules, the molecules are expressedby any means known in the art. In one variation the molecules are clonedinto expression vectors, which are in turn used to transform ortransfect cells to express the polypeptides. In another variation, thepolynucleotides are cloned into a phage display vector system forscreening. [See, e.g., Chang et al. (1999).] The screening assay mayentail a direct receptor binding assay as described below in Example 3.Alternatively, receptor binding may be assayed indirectly by assayingfor a biological activity induced by receptor binding. Thus, in onevariation, the screening step comprises contacting the hybridpolypeptide to a cell that expresses the receptor, wherein changes incell growth or cell survival induced by the hybrid polypeptide isindicative of binding between the hybrid polypeptide and the receptor.

In a preferred variation of the method, the screening and selectingsteps are designed to select polynucleotides that encode polypeptidesthat have novel receptor binding profiles not possessed by the naturallyoccurring VEGFs from which the polypeptide was derived. For example, themethod is practiced wherein the screening step comprises screening toidentify a hybrid polypeptide that binds human VEGFR-1 and humanVEGFR-3, and the selecting step comprises selecting a hybrid polypeptidethat binds human VEGFR-1 and human VEGFR-3, but fails to bind humanVEGFR-2. Alternatively, the method is practiced whereby the screeningstep comprises screening to identify a hybrid polypeptide that bindshuman VEGFR-1, VEGFR-2, and human VEGFR-3, and the selecting stepcomprises selecting a hybrid polypeptide that binds human VEGFR-1,VEGFR-2, and human VEGFR-3.

In a related embodiment, the invention provides a method for making apolynucleotide that encodes a polypeptide that modulates the growth ofmammalian endothelial cells, comprising the steps of: (a) preparing aset of polynucleotide fragments having the following characteristics:(i) the set includes a first subset of coding polynucleotide fragments,wherein each coding polynucleotide fragment of the first subset encodesat least four amino acids of the amino acid sequence of a firstmammalian vascular endothelial growth factor; (ii) the set includes asecond subset of coding polynucleotide fragments, wherein each codingpolynucleotide fragment of the second subset encodes at least four aminoacids of the amino acid sequence of a second mammalian vascularendothelial growth factor; (b) commingling the polynucleotide fragmentswhich comprise the set under conditions wherein the codingpolynucleotide fragments from the first and second subsets recombine toform hybrid polynucleotides; (c) expressing the hybrid polynucleotidesto make hybrid polypeptides encoded by the hybrid polynucleotides; (d)screening the hybrid polypeptides to identify a hybrid polypeptide thatmodulates the growth of mammalian endothelial cells; and (e) selectingthe polynucleotide that encodes the hybrid polypeptide that modulatesthe growth of mammalian endothelial cells in the screening step.

Practice of these methods of generating hybrid polynucleotides usingmammalian vascular endothelial growth factors that comprise a receptorbinding domain characterized by eight cysteines that are conserved inhuman Vascular Endothelial Growth Factor A (VEGF-A), human VascularEndothelial Growth Factor B (VEGF-B), human Vascular Endothelial GrowthFactor C (VEGF-C), and human Vascular Endothelial Growth Factor D(VEGF-D) is preferred. Exemplary starting molecules include VEGF-AVEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF, PDGF-A, and PDGF-B polypeptides ofhuman and other mammals. Also included is the recently describedprotein, fallotein, disclosed in the EMBL database (Acc. No. AF091434)(SEQ ID NO: 149), which has structural characteristics of the PDGF/VEGFfamily of growth factors. Thus, it is also contemplated to use falloteinin generating hybrid proteins together with other mammalian VEGFs.

The polynucleotide and encoded polypeptide products of the foregoingmethods are themselves considered to be additional aspects of thepresent invention.

Antibodies that may be generated against polypeptides of the invention,and that bind polypeptides of the invention with an affinity greaterthan for any natural occurring VEGF, also are contemplated as aspects ofthe invention. Polypeptides comprising the antigen-binding fragments ofsuch antibodies also are contemplated as an aspect of the invention.Antibodies that bind to the polypeptides of the invention but not tovertebrate VEGF's are contemplated.

In yet another embodiment, the invention provides numerous in vitro andin vivo methods of using polypeptides and polynucleotides of theinvention. Such methods are described in greater detail below in theDetailed Description. Generally speaking, polypeptides of the inventionare useful for modulating (stimulating or inhibiting) cellular processesthat are mediated through any of the PDGF/VEGF family of receptors, suchas PDGFR-α, PDGFR-β, VEGFR-1, VEGFR-2, and/or VEGFR-3. These receptorsmay be involved singularly in certain processes and in combination, tovarying extents, in other processes. Polypeptides of the inventionpossess many different receptor binding profiles, and one of theadvantages of the invention is the ability to select a polypeptide witha receptor binding profile that matches the receptor expression profileof the biological process to be modulated.

Thus, in one variation, the invention provides a method of modulatingthe signaling of one or more of PDGFR-α, PDGFR-β, VEGFR-1, VEGFR-2,and/or VEGFR-3 in a cell, comprising the step of contacting a cell thatexpresses one or more of PDGFR-α, PDGFR-β, VEGFR-1, VEGFR-2, and/orVEGFR-3 with a composition comprising a polypeptide of the invention. Inone variation, modulation to activate signaling is contemplated, and thecell is contacted with a polypeptide of the invention that stimulatesreceptor signaling in an amount sufficient to bind to the one or morereceptors and induce receptor signaling. In another variation,modulation to inhibit signaling is contemplated. The cell is contactedwith a polypeptide that inhibits ligand-induced receptor activation (ora polypeptide conjugated to a cytotoxin), in an amount sufficient toinhibit signaling that is induced by receptor ligand growth factorpolypeptides that exist endogenously in the cell's environment.Dose-response studies permit accurate determination of a proper quantityof polypeptide to employ. Effective quantities can be estimated frommeasurements of the binding affinity of a polypeptide for a targetreceptor, of the quantity of receptor present on target cells, of theexpected dilution volume (e.g., patient weight and blood volume for invivo embodiments), and of polypeptide clearance rates.

In another variation, the invention provides a method of modulating thesignaling of one or more of PDGFR-α, PDGFR-β, VEGFR-1, VEGFR-2, and/orVEGFR-3, comprising the step or administering to a patient in need ofmodulation of the signaling of one or more of these receptors acomposition comprising a polynucleotide of the invention, underconditions in which cells of the patient are transformed or transfectedby the polynucleotide and express the polypeptide of the inventionencoded thereby, wherein the expressed polypeptide modulates signalingof the one or more receptors.

As discussed below, analysis of the chimeras receptor binding propertiesand the sequences of VEGFR-3 ligands in relation to the sequence ofVEGF-A suggests that Fragments 4 and 5 from the VEGF-C molecule areimportant for conferring VEGFR-3 binding affinity, and in particular thesequence of residues TNTFxxxP (SEQ ID NO: 1204) found within Fragments 4and 5. Thus, in another variation, the invention provides moleculesdesigned using these core residues and other substituents to modulateVEGFR-3 biological activity. For example, in one embodiment, theinvention provides a molecule comprising the peptide sequence TNTFX_(n)P(SEQ ID NO: 1212), wherein X_(n) comprises from one to seven aminoacids, and wherein the molecule inhibits VEGF-C-mediated activation ofVEGFR-3. The molecule may include additional residues or organicmoieties. In one variation, it is contemplated that this epitope will belinked by a non-VEGF-C amino acid sequence to other epitopes involved inreceptor binding, thereby creating a molecule capable of interactingwith receptor loci involved in ligand binding and blockingligand-mediated activation of the receptor. In a preferred embodiment,X_(n) comprises three amino acids, which represents the same amino acidspacing as native VEGF-C.

In a related embodiment, the invention provides a molecule comprisingthe human VEGF-C peptide sequence EFGVATNTFFKPPCVSVYRCG (SEQ ID NO:1205) or a fragment or variant thereof, wherein the molecule inhibitsVEGF-C-mediated activation of VEGFR-3. In one variation, the fragment issuch that the molecule comprises the amino acid sequenceEFGVATNTFFKPPCVSVYRCG (SEQ ID NO: 1205). In another variation, thefragment is such that the molecule comprises the amino acid sequenceTNTFFKPP (SEQ ID NO: 1206). In still another variation, the fragment orvariant comprises the amino acid sequence TNTFFKPPCVxxxR (SEQ ID NO:1207), or the amino acid sequence TNTFFKPPCVxxxRCGGCC (SEQ ID NO: 1208).

Data relating to binding properties and sequence of chimeric moleculesof the invention also provides insight into the important amino acidtargets for synthetic design of modulators of receptor/ligandinteractions. For example, in one embodiment, the invention provides amethod for identifying a modulator of VEGFR-1 binding to VEGF-Acomprising the steps of (i) measuring binding between VEGFR-1 and VEGF-Ain the presence and absence of a test compound under conditions thatallow binding of VEGFR-1 to VEGF-A, and (ii) identifying as a modulatora test compound which alters VEGFR-1 binding to VEGF-A and which bindsVEGF-A at a site defined by Phe⁴³, Met⁴⁴, Tyr⁴⁷, Gln⁴⁸, Tyr⁵¹, Gln¹⁰⁵,and Met¹⁰⁷ of SEQ ID NO: 2, or which binds VEGFR-1 at VEGFR-1 residueswhich interface with said residues of SEQ ID NO: 2. Modulators that actas inhibitors, and are useful for ameliorating conditions characterizedby undesirable or excessive ligand-mediated receptor activation, are apreferred class of modulators. Activators are another preferred class.

In a related embodiment, the invention provides a method for identifyinga modulator of VEGFR-1 binding to VEGF-A comprising the steps of (i)measuring binding between VEGFR-1 and VEGF-A in the presence and absenceof a test compound under conditions that allow binding of VEGFR-1 toVEGF-A, and (ii) identifying as a modulator a test compound which altersVEGFR-1 binding to VEGF-A and which binds VEGF-A at a site defined byLys⁴², Phe⁴³, Met⁴⁴, Tyr⁴⁷, Gln⁴⁸, Tyr⁵¹, Ile⁷², Lys⁷⁴, Asp⁸⁹, Gly⁹¹,Leu⁹², Gln¹⁰⁵, Met¹⁰⁷, Ile¹⁰⁹, Phe¹¹¹, His¹¹², Gln¹¹⁵, Ile¹¹⁷, Glu¹²⁹,Arg¹³¹, and Pro¹³² of SEQ ID NO: 2, or which binds VEGFR-1 at VEGFR-1residues which interface with said residues of SEQ ID NO: 2.

Similarly, the invention provides a method for identifying a modulatorof VEGFR-3 binding to VEGF-C comprising the steps of (i) measuringbinding between VEGFR-3 and VEGF-C in the presence and absence of a testcompound under conditions that allow binding of VEGFR-3 to VEGF-C, and(ii) identifying as a modulator a test compound which alters VEGFR-3binding to VEGF-C and which binds VEGF-C at a site defined by Lys¹²⁰,Ser¹²¹, Ile¹²², Trp¹²⁶, Arg¹²⁷, Gln¹³⁰, Phe¹⁵¹, Lys¹⁵³, Ser¹⁶⁸, Gly¹⁷⁰,Leu¹⁷¹, Tyr¹⁸⁴, Phe¹⁸⁶, Ile¹⁹⁰, Pro¹⁹¹, Pro¹⁹⁶, Pro¹⁹⁸, Arg²¹⁰, Met²¹²,and Ser²¹³ of SEQ ID NO: 22, or which binds VEGFR-3 at VEGFR-3 residueswhich interface with said residues of SEQ ID NO: 22. The invention alsoprovides a method for identifying a modulator of VEGFR-3 binding toVEGF-C comprising the steps of (i) measuring binding between VEGFR-3 andVEGF-C in the presence and absence of a test compound under conditionsthat allow binding of VEGFR-3 to VEGF-C, and (ii) identifying as amodulator a test compound which alters VEGFR-3 binding to VEGF-C andwhich binds VEGF-C at a site defined by Thr¹⁴⁸, Asn¹⁴⁹, Thr¹⁵⁰, Phe¹⁵¹,and Pro¹⁵⁵ of SEQ ID NO: 22, or which binds VEGFR-3 at VEGFR-3 residueswhich interface with said residues of SEQ ID NO: 22.

Also contemplated as aspects of the invention are compositions thatcomprise modulators identified by the foregoing methods, especiallycompositions comprising substantially purified modulators in apharmaceutically acceptable carrier. Similarly, use of such modulatorsin the manufacture of a medicament for the treatment of disease statescharacterized by abnormal vascular endothelial growth factor receptoractivity is contemplated.

In still another variation, any of the foregoing methods optionallyinclude the additional step of administering the identified modulator toa patient in need of treatment for a disease state characterized byundesirable levels of receptor activity; or a step of contacting cellsthat express the receptor to modulate the level of receptor activity inthe cells.

Additional embodiments, features, and variations of the invention willbe apparent to those skilled in the art from the entirety of thisapplication, including the Drawing and the Detailed Description, and allsuch features are intended as aspects of the invention.

Likewise, features of the invention described herein can be re-combinedinto additional embodiments that also are intended as aspects of theinvention, irrespective of whether the combination of features isspecifically mentioned above as an aspect or embodiment of theinvention. Also, only such limitations which are described herein ascritical to the invention should be viewed as such; variations of theinvention lacking limitations which have not been described herein ascritical are intended as aspects of the invention.

With respect to aspects of the invention that have been described as aset or genus, every individual member of the set or genus is intended,individually, as an aspect of the invention.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically mentioned above. Although theapplicant(s) invented the full scope of the claims appended hereto, theclaims appended hereto are not intended to encompass within their scopethe prior art work of others. Therefore, in the event that statutoryprior art within the scope of a claim is brought to the attention of theapplicants by a Patent Office or other entity or individual, theapplicant(s) reserve the right to exercise amendment rights underapplicable patent laws to redefine the subject matter of such a claim tospecifically exclude such statutory prior art or obvious variations ofstatutory prior art from the scope of such a claim. Variations of theinvention defined by such amended claims also are intended as aspects ofthe invention.

BRIEF DESCRIPTION OF THE DRAWING

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Patent and Trademark Office uponrequest and payment of the necessary fee.

FIG. 1 depicts a perspective view of a three-dimensional model of aVEGF-A monomer, in which selected secondary structure elements areidentified. A VEGF-A-encoding polynucleotide was divided into ninesegments for construction of VEGF-A/VEGF-C chimeras, and labels 1-9identify the location of the peptides encoded by each of the ninesegments.

FIG. 2 is a schematic diagram of the 9 VEGF-A and 9 VEGF-C DNA fragmentsused to construct the VEGF-A/VEGF-C hybrid molecules. These fragmentsare numbered 1 through 9 on top. The N123, N45, C67 and C89 groups offragments are also indicated. N123 consists of 3 VEGF-A fragments and 3VEGF-C fragments (fragments 1-3), whereas N45 (fragments 4-5), C67(fragments 6-7), and C89 (fragments 8-9) each consist of 2 VEGF-Afragments and 2 VEGF-C fragments. Selected restriction endonucleasesites also are depicted.

FIG. 3 schematically depicts all 8 possible DNA molecules correspondingto the N123 region, resulting from ligating different combinations offragments 1, 2, and 3 from VEGF-A (A1, A2, and A3) and VEGF-C (C1, C2,and C3).

FIG. 4 schematically depicts all 4 possible DNA molecules correspondingto the N45 region resulting from ligating different combinations offragments 4 and 5 from VEGF-A and VEGF-C.

FIG. 5 schematically depicts all 4 possible DNA molecules correspondingto the C67 region resulting from ligating different combinations offragments 6 and 7 from VEGF-A and VEGF-C.

FIG. 6 schematically depicts all 4 possible DNA molecules correspondingto the C89 region resulting from ligating different combinations offragments 8 and 9 from VEGF-A and VEGF-C.

FIGS. 7A-7D depict in boxes the amino acid sequences encoded by DNAfragments A1-A9 (SEQ ID NOs: 128-136) and C1-C9 (SEQ ID NOs: 137-145),and depict the manner in which longer encoded amino acid sequences wereformed through the joining of fragments A1-A3 and C1-C3 in the N123ligation (FIG. 7A); of fragments A4-A5 and C4-C5 in the N45 ligation(FIG. 7B); of fragments A6-A7 and C6-C7 in the C67 ligation; and offragments A8-A9 and C8-C9 in the C89 ligation. In each figure, arrowsrepresent peptide bonds between encoded amino acid sequences that resultfrom proper ligation of compatible DNA fragments and translation of theresultant ligated DNA.

FIG. 8 is a three-dimensional model showing the interaction of a VEGF-Adimer with two VEGFR-1 molecules. The two VEGF-A monomers are colored ingreen and blue, respectively. The two VEGFR-1 receptors are depicted ingray. Red represents the location of residues within the VEGF-A monomersimportant for interfacing with VEGFR-1. These residues are clustered atthe two ends of the VEGF-A dimer and include the N-terminal helix andpart of the β5 strand.

FIG. 9 is a three-dimensional model depicting the groove formed by aVEGF-C dimer. The entry and the sides of this groove are formed by thefragments, described in Example 4, that appear to be important forconferring VEGFR-3 specificity. The green and blue indicate the twoVEGF-C monomers and the gray indicates a VEGFR-3 receptor molecule. TheVEGF-C residues that participate in binding VEGFR-3 are indicated inyellow.

FIG. 10 is a three-dimensional model of a the interaction between aVEGF-C dimer and a single VEGFR-3 molecule, extrapolated from theVEGF-A/VEGFR-A model. Blue and green represent the two VEGF-C monomersand grey represents VEGFR-3. Fragment 5 of the green VEGF-C monomer isshown in orange and fragment 4 of the same monomer is shown in white.Residues in red are those located within fragment 4 or 5 that areprobably in contact with the receptor.

DETAILED DESCRIPTION

The present invention provides novel polypeptides that bind cellularreceptors for vascular endothelial growth factor polypeptides;polynucleotides encoding such polypeptides; compositions comprising thepolypeptides and polynucleotides; and methods and uses involving theforegoing. These materials and methods are described in detail in thepreceding Summary of Invention section, which is hereby incorporatedinto the Detailed Description in its entirety. Some polypeptides of theinvention exhibit unique receptor binding profiles compared to known,naturally occurring vascular endothelial growth factors.

Methods of Making Peptides

The peptides of the present invention may be synthesized using a varietyof methods, including those described in the summary of invention andthe examples. The peptides of the present invention can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co., (1984); Tam et al., J. Am. Chem. Soc., 105:6442, (1983);Merrifield, Science, 232: 341-347, (1986); and Barany and Merrifield,The Peptides, Gross and Meienhofer, eds, Academic Press, New York,1-284; Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987);and U.S. Pat. No. 5,424,398), each incorporated herein by reference.

Solid phase peptide synthesis methods use acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.These methods for peptide synthesis use butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxy-carbonyl (FMOC) protection of alpha-amino groups.Both methods involve stepwise syntheses whereby a single amino acid isadded at each step starting from the C-terminus of the peptide (See,Coligan, et al., Current Protocols in Immunology, Wiley Interscience,1991, Unit 9) On completion of chemical synthesis, the peptides can bedeprotected to remove the t-t-BOC or FMOC amino acid blocking groups andcleaved from the polymer by treatment with acid at reduced temperature(e.g., liquid HF-10% anisole for about 0.25 to about 1 hours at 0□C).After evaporation of the reagents, the peptides are extracted from thepolymer with 1% acetic acid solution which is then lyophilized to yieldthe crude material. This can normally be purified by such techniques asgel filtration on Sephadex G-15 using 5% acetic acid as a solvent.Lyophilization of appropriate fractions of the column will yield thehomogeneous peptide or peptide derivatives, which can then becharacterized by such standard techniques as amino acid analysis, thinlayer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopy, molar rotation, solubility, andquantitated by the solid phase Edman degradation.

Other methods, such as selecting peptides from a phage display library,are available for improving upon peptide specifically described herein.Libraries can be prepared from sets of amino acids as described herein.Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. ml 13, fd, or lambda phage), displayinginserts from 4 to about 80 amino acid residues. The inserts mayrepresent, for example, a completely degenerate or biased array. Onethen can select phage-bearing inserts which bind to the target VEGFreceptor(s). This process can be repeated through several cycles ofreselection of phage that bind to the target receptor(s). Repeatedrounds lead to enrichment of phage bearing particular sequences. DNAsequence analysis can be conducted to identify the sequences of theexpressed polypeptides. The minimal linear portion of the sequence thatbinds to the target receptor(s) can be determined. One can repeat theprocedure using a biased library containing inserts containing part orall of the minimal linear portion plus one or more additional degenerateresidues upstream or downstream thereof. These techniques may identifypeptides of the invention with still greater receptor binding affinitythan peptides already identified herein. Screening resultant peptideagainst multiple receptors will identify peptides with multiple receptorbinding affinities. Yeast two-hybrid screening methods also may be usedto identify polypeptides that bind to the target receptor(s).

Alternatively, a variety of expression vector/host systems may beutilized to contain and express the chimeric peptides of the presentinvention. These include but are not limited to microorganisms such asbacteria transformed with recombinant bacteriophage, plasmid or cosmidDNA expression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems. Mammalian cells that are usefulin recombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of theprotein are described herein below.

For example, the chimeric peptide may be recombinantly expressed inyeast using a commercially available expression system, e.g., the PichiaExpression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol.

The secreted peptide is purified from the yeast growth medium by, e.g.,the methods used to purify the chimeric peptide from bacterial andmammalian cell supernatants.

Alternatively, the cDNA encoding the peptide may be cloned into thebaculovirus expression vector pVL1393 (PharMingen, San Diego, Calif.).This vector is then used according to the manufacturer's directions(PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-freemedia and to produce recombinant protein. The protein is purified andconcentrated from the media using a heparin-Sepharose column (Pharmacia,Piscataway, N.J.) and sequential molecular sizing columns (Amicon,Beverly, Mass.), and resuspended in PBS. SDS-PAGE analysis shows asingle band and confirms the size of the protein, and Edman sequencingon a Porton 2090 Peptide Sequencer confirms its N-terminal sequence.

Alternatively, the peptide may be expressed in an insect system. Insectsystems for protein expression are well known to those of skill in theart. In one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The peptide coding sequenceis cloned into a nonessential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of the peptide will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein coat. Therecombinant viruses are then used to infect S. frugiperda cells orTrichoplusia larvae in which peptide is expressed (Smith et al., J Virol46: 584, 1983; Engelhard E K et al., Proc Nat Acad Sci 91: 3224-7,1994).

In another example, the DNA sequence encoding the peptide is amplifiedby PCR and cloned into an appropriate vector for example, pGEX-3X(Pharmacia, Piscataway, N.J.). The pGEX vector is designed to produce afusion protein comprising glutathione-S-transferase (GST), encoded bythe vector, and a protein encoded by a DNA fragment inserted into thevector's cloning site. The primers for the PCR may be generated toinclude for example, an appropriate cleavage site.

Where the fusion partner was used solely to facilitate expression or isotherwise not desirable as an attachment to the peptide of interest, therecombinant fusion protein may then be cleaved from the GST portion ofthe fusion protein. The pGEX-3X/chimeric peptide construct istransformed into E. coli XL-1 Blue cells (Stratagene, La Jolla Calif.),and individual transformants were isolated and grown. Plasmid DNA fromindividual transformants is purified and partially sequenced using anautomated sequencer to confirm the presence of the desired chimericpeptide encoding nucleic acid insert in the proper orientation.

Particularly preferred peptide compositions of the present invention arethose which are conjugated to any anti-tumor peptide such as a tumornecrosis factor (TNF). In a particularly preferred method, theTNF-peptides chimeras are generated as recombinant fusions withpeptide-encoding sequences fused in frame to TNF (Novagen) encodingsequences. Peptide-TNF cDNA is cloned into pET-11b vector (Novagen) andthe expression of TNF-peptides in BL21 E. coli is induced according tothe pET11b manufacturer's instruction. Soluble TNF-peptides are purifiedfrom bacterial lysates by ammonium sulfate preparation, hydrophobicinteraction chromatography on Phenyl-Sepharose 6 Fast Flow, ion exchangechromatography on DEAE-Sepharose Fast Flow and gel filtrationchromatography on Sephacryl-S-300 HR.

It is contemplated that recombinant protein production also may be usedto produce the chimeric peptide compositions. For example, induction ofthe GST/chimeric peptide is achieved by growing the transformed XL-1Blue culture at 37□C in LB medium (supplemented with carbenicillin) toan optical density at wavelength 600 nm of 0.4, followed by furtherincubation for 4 hours in the presence of 0.5 mM Isopropylβ-D-Thiogalactopyranoside (Sigma Chemical Co., St. Louis Mo.).

The fusion protein, expected to be produced as an insoluble inclusionbody in the bacteria, may be purified as follows. Cells are harvested bycentrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; andtreated with 0.1 mg/ml lysozyme (Sigma Chemical Co.) for 15 minutes atroom temperature. The lysate is cleared by sonication, and cell debrisis pelleted by centrifugation for 10 minutes at 12,000×g. The fusionprotein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mMEDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000×g.The pellet is resuspended in standard phosphate buffered saline solution(PBS) free of Mg⁺⁺ and Ca⁺⁺. The fusion protein is further purified byfractionating the resuspended pellet in a denaturing SDS polyacrylamidegel (Sambrook et al., supra). The gel is soaked in 0.4 M KCl tovisualize the protein, which is excised and electroeluted in gel-runningbuffer lacking SDS. If the GST/chimeric peptide fusion protein isproduced in bacteria as a soluble protein, it may be purified using theGST Purification Module (Pharmacia Biotech).

The fusion protein may be subjected to digestion to cleave the GST fromthe chimeric peptide of the invention. The digestion reaction (20-40 μgfusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mlPBS) is incubated 16-48 hrs. at room temperature and loaded on adenaturing SDS-PAGE gel to fractionate the reaction products. The gel issoaked in 0.4 M KCl to visualize the protein bands. The identity of theprotein band corresponding to the expected molecular weight of chimericpeptide may be confirmed by amino acid sequence analysis using anautomated sequencer (Applied Biosystems Model 473A, Foster City,Calif.). Alternatively, the identity may be confirmed by performing HPLCand/or mass spectometry of the peptides.

Alternatively, the DNA sequence encoding the chimeric peptide may becloned into a plasmid containing a desired promoter and, optionally, aleader sequence (see, e.g., Better et al., Science, 240:1041-43, 1988).The sequence of this construct may be confirmed by automated sequencing.The plasmid is then transformed into E. coli strain MC1061 usingstandard procedures employing CaCl2 incubation and heat shock treatmentof the bacteria (Sambrook et al., supra). The transformed bacteria aregrown in LB medium supplemented with carbenicillin, and production ofthe expressed protein is induced by growth in a suitable medium. Ifpresent, the leader sequence will effect secretion of the chimericpeptide and be cleaved during secretion.

The secreted recombinant protein is purified from the bacterial culturemedia by the method described herein below.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art. Host cell strains maybe chosen for a particular ability to process the expressed protein orproduce certain post-translation modifications that will be useful inproviding protein activity. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation. Different hostcells such as CHO, HeLa, MDCK, 293, W138, and the like have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

It is preferable that the transformed cells are used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside G418; also that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, beta-glucuronidase and its substrate, GUS, and luciferaseand its substrate, luciferin.

For certain applications, it may be desirable to produce peptides orpolypeptides of the present invention which are resistant to proteolyticdigestion. Such peptides may include non-hydrolyzable peptide bonds, andpeptides having end modifications such as an amide (e.g., CONH₂) at theC-terminus or a acetyl group at the N-terminus. It is contemplated thatthe peptides of the invention are modified such that their in vivo halflife is increased, their physical stability is increased, rate of invivo release and rate of in vivo clearance also may be affected.

To prepare non-hydrolyzable peptides, one may select peptides from alibrary non-hydrolyzable peptides, or introduce modifications to selectpeptides, such as one or more D-amino acids or one or morenon-hydrolyzable peptide bonds linking amino acids. For example, one canselect peptides having a desired receptor binding profile and thenmodify such peptides as necessary to reduce the potential for hydrolysisby proteases. For example, to determine the susceptibility toproteolytic cleavage, peptides may be labeled and incubated with cellextracts or purified proteases and then isolated to determine whichpeptide bonds are susceptible to proteolysis, e.g., by sequencingpeptides and proteolytic fragments. Alternatively, potentiallysusceptible peptide bonds can be identified by comparing the amino acidsequence of the peptides of the present invention with the knowncleavage site specificity of a panel of proteases. Based on the resultsof such assays, individual peptide bonds which are susceptible toproteolysis can be replaced with non-hydrolyzable peptide bonds by invitro synthesis of the peptide.

Many non-hydrolyzable peptide bonds are known in the art, along withprocedures for synthesis of peptides containing such bonds.Non-hydrolyzable bonds include —[CH₂NH]— reduced amide peptide bonds,—[COCH₂]— ketomethylene peptide bonds, —[CH(CN)NH]—(cyanomethylene)amino peptide bonds, —[CH₂CH(OH)]— hydroxyethylenepeptide bonds, —[CH₂O]— peptide bonds, and —[CH₂S]— thiomethylenepeptide bonds (see e.g., U.S. Pat. No. 6,172,043).

Peptides useful in the invention can be linear, or may be circular orcyclized by natural or synthetic means. For example, disulfide bondsbetween cysteine residues may cyclize a peptide sequence. Bifunctionalreagents can be used to provide a linkage between two or more aminoacids of a peptide. Other methods for cyclization of peptides, such asthose described by Anwer et al. (Int. J. Pep. Protein Res. 36:392-399,1990) and Rivera-Baeza et al. (Neuropeptides 30:327-333, 1996) are alsoknown in the art.

Furthermore, nonpeptide analogs of peptides which provide a stabilizedstructure or lessened biodegradation, are also contemplated. Peptidemimetic analogs can be prepared based on a selected peptide byreplacement of one or more residues by nonpeptide moieties. Preferably,the nonpeptide moieties permit the peptide to retain its naturalconfirmation, or stabilize a preferred, e.g., bioactive, confirmation.One example of methods for preparation of nonpeptide mimetic analogsfrom peptides is described in Nachman et al., Regul. Pept. 57:359-370(1995). Peptide as used herein embraces all of the foregoing.

The polypeptides of the invention include polypeptides that aremodified, for instance, by glycosylation, amidation, carboxylation, orphosphorylation, or by the creation of acid addition salts, amides,esters, in particular C-terminal esters, and N-acyl derivatives.

Also, as described above, the invention embraces polypeptides modifiedby forming covalent or noncovalent complexes with other moieties.Covalently-bound complexes can be prepared by linking the chemicalmoieties to functional groups on the side chains of amino acidscomprising the peptides, or at the N- or C-terminus.

In particular, it is anticipated that the aforementioned peptides can beconjugated to a reporter group, including, but not limited to aradiolabel, a fluorescent label, an enzyme (e.g., that catalyzes acolorimetric or fluorometric reaction), a substrate, a solid matrix, ora carrier (e.g., biotin or avidin). The invention accordingly provides amolecule comprising a chimeric polypeptide comprising a plurality ofpeptide subunits derived from two or more vascular endothelial growthfactor polypeptides, wherein the chimeric polypeptide preferably furthercomprises a reporter group selected from the group consisting of aradiolabel, a fluorescent label, an enzyme, a substrate, a solid matrix,and a carrier. The use of such labels is well known and is described in,e.g., U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No.3,996,345 and U.S. Pat. No. 4,277,437. Other labels that will be usefulinclude but are not limited to radioactive labels, fluorescent labelsand chemiluminescent labels. U.S. patents concerning use of such labelsinclude for example U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752;U.S. Pat. No. 3,939,350 and U.S. Pat. No. 3,996,345. Any of the peptidesof the present invention may comprise one, two, or more of any of theselabels.

Methods of Using the Polypeptides of the Invention

The many biological activities mediated through the PDGF/VEGF receptorfamily (including but not limited to affecting growth and migration ofvascular endothelial cells and blood vessels; promoting growth oflymphatic endothelial cells and lymphatic vessels; increasing vascularpermeability; and affecting myelopoiesis) support numerous diagnosticand in vitro and in vivo clinical utilities for polypeptides of theinvention that are capable of binding one of more members of the VEGFreceptor family, for modulating (stimulating or inhibiting) thesebiological activities.

Multiple mechanisms exist through which polypeptides of the inventionwill act as growth factors (i.e., agonists or receptor stimulants). Forexample, polypeptides of the invention that form homodimers that bindand activate one or more members of the VEGF receptor family will beuseful as vascular endothelial growth factors. Alternatively,polypeptides of the invention that form heterodimers with endogenousgrowth factor polypeptides (VEGF-A or VEGF-C or other family members)will also be effective agonists, provided that the heterodimers soformed are capable of binding and activating receptors to induce signaltransduction.

Multiple mechanisms exist through which polypeptides of the inventionwill act as inhibitors (antagonists) of growth factors of the VEGFfamily. Polypeptides of the invention that bind but fail to stimulateone or more receptors will inhibit stimulation of the receptor byendogenous growth factor, thereby acting as an inhibitor of endogenousgrowth factor. Such failure to stimulate may be due, in whole or inpart, to an inability to dimerize the receptor, perhaps due to aninability of the hybrid polypeptide of the invention to form growthfactor homodimers. Polypeptides of the invention that form heterodimerswith endogenous growth factor polypeptides will inhibit stimulation ofVEGF receptors if the heterodimer fails to bind receptors, or if theheterodimer binds only to an individual receptor or a heterologousreceptor pair in a manner that prevents receptor activation and signaltransduction. Whichever the mechanism, polypeptides of the inventionthat form activity-destroying heterodimers with endogenous VEGFpolypeptides (and that do not form active homodimers) are useful asantagonists of natural endogenous VEGF activity. Also, any polypeptidethat binds a receptor can be conjugated to a cytotoxic or cytostaticagent in order to deliver such agents to target cells. The attachment ofsuch agent is another means for inhibiting growth of cells in which VEGFpolypeptides exhibit a mitogenic response. Exemplary toxins includechemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.

It also will be apparent that two or more hybrid polypeptides of theinvention can be mixed, and that heterodimers so formed will be usefulas modulators depending upon their receptor binding and stimulatingproperties. Because polypeptides of the invention are hybrids derivedfrom naturally-occurring vascular endothelial growth factors that mayhave different receptor binding profiles, it is contemplated that someof the hybrids will act as activators of one or more receptors, and somewill act as inhibitors of one or more receptors. Procedures describedherein and other procedures known in the art can be used to determinereceptor binding, receptor activation, and receptor inhibitionproperties of polypeptides of the invention.

The polypeptides of the invention that bind and activate one or moreVEGF receptors may be useful for promoting angiogenesis and/orlymphangiogenesis, for example, to promote wound healing, to facilitatetissue transplantation, and to promote the formation of collateralvessels around arterial stenoses, and into injured tissues afterinfarction, to treat ischemia. On the other hand, polypeptides of theinvention that behave as antagonists of endogenous VEGF proteins can beused in therapeutic applications to treat diseases such as neoplasias,retinopathy, rheumatoid arthritis, and psoriasis, in which suppressionof angiogenesis is desirable.

Polypeptides of the present invention differ from natural VEGF receptorligands in that some of them selectively bind one of the VEGF receptorsand can thus be used to specifically induce signaling through oneparticular VEGF receptor. For example, polypeptides that solely induceVEGFR-3 signaling can be used therapeutically to target the lymphaticendothelia of individuals affected with lymphatic disorders, to improvethe structure and function of the lymphatic vasculature of suchindividuals. Such polypeptides also can be used to target neoplasiacharacterized by cells expressing VEGFR-3 on their surfaces. Chemotaxisof monocytes/macrophages [Barleon et al., Blood 87:3336-3343 (1996)] dueto VEGFs is mediated by VEGFR-1. Thus, molecules that specificallytarget the VEGFR-1 receptor can be used to direct therapeutic effects onthis particular VEGF receptor. For example, inhibitors of VEGFR-1 may beused to prevent virally induced angiogenesis, and molecules thatspecifically activate VEGFR-1 can be used to enhance monocyte/macrophagemigration. VEGFR-2 is essential for angiogenesis and sufficient forvirally-induced angiogenesis. Thus, inhibitors of VEGFR-2 may be usedfor inhibiting angiogenesis, including that induced by viral VEGFs,whereas molecules that stimulate VEGFR-2 can be useful for promotingangiogenesis.

A subset of the polypeptides of the present invention can bindcombinations of VEGF receptors not demonstrated for known natural VEGFligands, or are able to bind all three known VEGF receptors VEGFR-1,R-2, and R-3. These polypeptides may be useful for therapies in whichthe activation or inhibition of different combinations of VEGF receptorsis desired.

Polypeptides of the invention that can activate VEGFR-3 can be used topromote the endothelial functions of lymphatic vessels and tissues suchas to treat loss of lymphatic vessels, occlusions of lymphatic vessels,lymphangiomas, and primary idiopathic lymphedemas, including Milroy'sdisease and lymphedema praecox, as well as secondary lymphedemas,including those resulting from removal of lymph nodes and vessels,radiotherapy and surgery in treatment of cancer, trauma and infection.Polynucleotides or polypeptides of the invention could be administeredpurely as a prophylacetic treatment to prevent lymphedema in subjects atrisk for developing lymphedema, or as a therapeutic treatment tosubjects afflicted with lymphedema, for the purpose of ameliorating itssymptoms (e.g., swelling due to the accumulation of lymph).

The polynucleotides and polypeptides of the invention that activateVEGFR-3 can also be used to promote re-growth or permeability oflymphatic vessels in patients whose axillary lymphatic vessels wereremoved during surgical interventions in the treatment of cancer (e.g.,breast cancer). Polynucleotides and polypeptides of the invention can beused to treat vascularization in, for example, organ transplantpatients. A composition containing the polypeptide(s) of the inventionmay be directly applied to the isolated vessel segment prior to itsbeing grafted in vivo to minimize rejection of the transplanted materialand to stimulate vascularization of the transplanted materials.

Polypeptides of the invention that activate VEGF receptor activity maybe used to treat wounds, surgical incisions, sores, and otherindications where healing is reasonably expected to be promoted if theprocess of neovascularization can be induced and/or accelerated.

As explained in greater detail above and reported in the literature, theexpression of receptors for vascular endothelial growth factors havebeen observed in certain progenitor cells, such as hematopoieticprogenitor cells, and VEGF-C has been observed to have myelopoieticactivity. These observations provide an indication that polynucleotidesor polypeptides according to the invention may be used to treat orprevent inflammation, infection, or immune disorders by modulating theproliferation, differentiation and maturation, or migration of immunecells or hematopoietic cells. Polynucleotides or polypeptides accordingto the invention may also be useful to promote or inhibit trafficking ofleukocytes between tissues and lymphatic vessels and migration in andout of the thymus.

Polynucleotides and polypeptides of the invention can be used forstimulating myelopoiesis (especially growth of neutrophilicgranulocytes) or inhibiting it. Thus, the invention includes a methodfor modulating myelopoiesis in a mammalian subject comprisingadministering to a mammalian subject in need of modulation ofmyelopoiesis an amount of a polypeptide of the invention that iseffective to modulate myelopoiesis. In one embodiment, a mammaliansubject suffering from granulocytopenia is selected, and the methodcomprises administering to the subject an amount of a polypeptideeffective to stimulate myelopoiesis. In particular, a polypeptide of theinvention is administered in an amount effective to increase theneutrophil count in blood of the subject.

In a related embodiment, the invention includes a method of increasingthe number of neutrophils in the blood of a mammalian subject comprisingthe step of expressing in a cell in a subject in need of an increasednumber of blood neutrophils a DNA encoding a polynucleotide of theinvention that is able to activate signaling through VEGF receptors, theDNA operatively linked to a promoter or other control sequence thatpromotes expression of the DNA in the cell. Similarly, the inventionincludes a method of modulating the growth of neutrophilic granulocytesin vitro or in vivo comprising the step of contacting mammalian stemcells with a polypeptide of the invention in an amount effective tomodulate the growth of mammalian endothelial cells.

The invention also includes a method for modulating the growth of CD34+progenitor cells (especially hematopoietic progenitor cells andendothelial progenitor cells) in vitro or in vivo comprising the step ofcontacting mammalian CD34+ progenitor cells with a polypeptide of theinvention in an amount effective to modulate the growth of mammalianendothelial cells. For in vitro methods, CD34+ progenitor cells isolatedfrom cord blood or bone marrow are specifically contemplated. In vitroand in vivo methods of the invention for stimulating the growth of CD34+precursor cells also include methods wherein polypeptides of theinvention are employed together (simultaneously or sequentially) withother polypeptide factors for the purpose of modulatinghematopoiesis/myelopoiesis or endothelial cell proliferation. Such otherfactors include, but are not limited to colony stimulating factors(“CSFs,” e.g., granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), andgranulocyte-macrophage-CSF (GM-CSF)), interleukin-3 (IL-3, also calledmulti-colony stimulating factor), other interleukins, stem cell factor(SCF), other polypeptide factors, and their analogs that have beendescribed and are known in the art. See generally The Cytokine Handbook,Second Ed., Angus Thomson (editor), Academic Press (1996); Callard andGearing, The Cytokine Facts Book, Academic Press Inc. (1994); andCowling and Dexter, TIBTECH, 10(10):349-357 (1992). The use of apolypeptide of the invention as a progenitor cell or myelopoietic cellgrowth factor or co-factor with one or more of the foregoing factors maypotentiate previously unattainable myelopoietic effects and/orpotentiate previously attainable myelopoietic effects while using lessof the foregoing factors than would be necessary in the absence of apolypeptide of the invention.

Polynucleotides and polypeptides of the invention may also be used inthe treatment of lung disorders to improve blood circulation in the lungand/or gaseous exchange between the lungs and the blood stream; toimprove blood circulation to the heart and O₂ gas permeability in casesof cardiac insufficiency; to improve blood flow and gaseous exchange inchronic obstructive airway disease; and to treat conditions such ascongestive heart failure, involving accumulations of fluid in, forexample, the lung resulting from increases in vascular permeability, byexerting an offsetting effect on vascular permeability in order tocounteract the fluid accumulation.

Polynucleotides and polypeptides of the invention could be used to treatmalabsorptive syndromes in the intestinal tract as a result of its bloodcirculation increasing and vascular permeability increasing activities.

Polypeptides of the invention that bind but do not stimulate signalingthrough one or more of the VEGF receptors may be used to treat chronicinflammation caused by increased vascular permeability, retinopathyassociated with diabetes, rheumatoid arthritis and psoriasis.

Polynucleotides or polypeptides according to the invention that are ableto inhibit the function of one or more VEGF receptors can also be usedto treat edema, peripheral arterial disease, Kaposi's sarcoma, orabnormal retinal development in premature newborns.

In another embodiment, the invention provides a method for modulatingthe growth of endothelial cells in a mammalian subject comprising thesteps of exposing mammalian endothelial cells to a polypeptide accordingto the invention in an amount effective to modulate the growth of themammalian endothelial cells. In one embodiment, the modulation of growthis affected by using a polypeptide capable of stimulating tyrosinephosphorylation of VEGF receptors in a host cell expressing the VEGFreceptors. In modulating the growth of endothelial cells, the inventioncontemplates the modulation of endothelial cell-related disorders. In apreferred embodiment, the subject, and endothelial cells, are human. Theendothelial cells may be provided in vitro or in vivo, and they may becontained in a tissue graft. An effective amount of a polypeptide is anamount necessary to achieve a reproducible change in cell growth rate(as determined by microscopic or macroscopic visualization andestimation of cell doubling time, or nucleic acid synthesis assays).

Since angiogenesis and neovascularization are essential for tumorgrowth, inhibition of angiogenic activity can prevent further growth andeven lead to regression of solid tumors. Likewise inhibition oflymphangiogenesis may be instrumental in preventing metastases.Polynucleotides and polypeptides of the invention may be useful to treatneoplasias including sarcomas, melanomas, carcinomas, and gliomas byinhibiting tumor angiogenesis.

Thus, it is contemplated that a wide variety of cancers may be treatedusing the peptides of the present invention including cancers of thebrain (glioblastoma, astrocytoma, oligodendroglioma, ependymomas), lung,liver, spleen, kidney, lymph node, pancreas, small intestine, bloodcells, colon, stomach, breast, endometrium, prostate, testicle, ovary,skin, head and neck, esophagus, bone marrow, blood or other tissue.

In many contexts, it is not necessary that the tumor cell be killed orinduced to undergo normal cell death or “apoptosis.” Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree or localized to a specific areaand inhibited from spread to disparate sites. It may be that the tumorgrowth is completely blocked, however, or that some tumor regression isachieved. Clinical terminology such as “remission” and “reduction oftumor” burden also are contemplated given their normal usage. In thecontext of the present invention, the therapeutic effect may result froman inhibition of angiogenesis and/or an inhibition of lymphangiogenesis.

Thus, the invention includes a method of treating a mammalian organismsuffering from a neoplastic disease characterized by expression of oneor more VEGF receptor(s) in cells, comprising the steps of: identifyinga mammalian organism suffering from a neoplastic disease statecharacterized by expression of VEGF receptor(s), and administering tothe mammalian organism in need of such treatment a composition, thecomposition comprising one or more polynucleotide(s) or polypeptide(s)of the invention effective to inhibit VEGF receptor-mediatedproliferation of the cells. Such treatment methodologies areparticularly indicated for neoplastic disease states that arecharacterized by neovascularization involving vessels lined withendothelial cells that express increased levels of one or more VEGFreceptors, relative to endothelial cells lining quiescent vessels; anddisease states characterized by a cancer cells that express VEGFreceptors. Targeting VEGFR-3 in tumor imaging and anti-tumor therapy isdescribed in PCT/US99/23525 (WO 00/21560), published 20 Apr. 2000,incorporated herein by reference. Other VEGF receptors (e.g., VEGFR-1)also have been implicated in tumor angiogenesis or metastasis.

Evidence exists that at least VEGF-C and VEGF-D of the VEGF family ofgrowth factors have utility for preventing stenosis or restenosis ofblood vessels. See International Patent Application No. PCT/US99/24054(WO 00/24412), “Use of VEGF-C or VEGF-D Gene or Protein to PreventRestenosis,” filed Oct. 26, 1999, incorporated herein by reference inits entirety. Polypeptides and polynucleotides of the invention alsowill have utility for these indications. Thus, in another aspect, theinvention provides a method of treating a mammalian subject to preventstenosis or restenosis of a blood vessel, comprising the step ofadministering to a mammalian subject in need of treatment to preventstenosis or restenosis of a blood vessel a composition comprising one ormore polypeptide(s) of the invention, in an amount effective to preventstenosis or restenosis of the blood vessel. In a preferred embodiment,the administering comprises implanting an intravascular stent in themammalian subject, where the stent is coated or impregnated with thecomposition. Exemplary materials for constructing a drug-coated ordrug-impregnated stent are described in literature cited above andreviewed in Lincoff et al., Circulation, 90: 2070-2084 (1994). Inanother preferred embodiment, the composition comprises microparticlescomposed of biodegradable polymers such as PGLA, non-degradablepolymers, or biological polymers (e.g., starch) which particlesencapsulate or are impregnated by a polypeptide(s) of the invention.Such particles are delivered to the intravascular wall using, e.g., aninfusion angioplasty catheter. Other techniques for achieving locallysustained drug delivery are reviewed in Wilensky et al., TrendsCardiovasc. Med., 3:163-170 (1993), incorporated herein by reference.

Administration via one or more intravenous injections subsequent to theangioplasty or bypass procedure also is contemplated. Localization ofthe polypeptides of the invention to the site of the procedure occursdue to expression of VEGF receptors on proliferating endothelial cells.Localization is further facilitated by recombinantly expressing thepolypeptides of the invention as a fusion polypeptide (e.g., fused to anapolipoprotein B-100 oligopeptide as described in Shih et al., Proc.Nat'l. Acad. Sci. USA, 87:1436-1440 (1990). Co-administration ofpolynucleotides and polypeptides of the invention is also contemplated.

Likewise, the invention also provides surgical devices that are used totreat circulatory disorders, such as intravascular or endovascularstents, balloon catheters, infusion-perfusion catheters, extravascularcollars, elastomeric membranes, and the like, which have been improvedby coating with, impregnating with, adhering to, or encapsulating withinthe device a composition comprising a polynucleotide or polypeptide ofthe invention.

Polynucleotides or polypeptides of the invention could be administeredpurely as a prophylacetic treatment to prevent stenosis, or shortlybefore, and/or concurrently with, and/or shortly after a percutaneoustransluminal coronary angioplasty procedure, for the purpose ofpreventing restenosis of the subject vessel. In another preferredembodiment, the polynucleotide or polypeptide is administered before,during, and/or shortly after a bypass procedure (e.g., a coronary bypassprocedure), to prevent stenosis or restenosis in or near thetransplanted (grafted) vessel, especially stenosis at the location ofthe graft itself. In yet another embodiment, the polynucleotide orpolypeptide is administered before, during, or after a vasculartransplantation in the vascular periphery that has been performed totreat peripheral ischemia or intermittent claudication. By prevention ofstenosis or restenosis is meant prophylacetic treatment to reduce theamount/severity of, and/or substantially eliminate, the stenosis orrestenosis that frequently occurs in such surgical procedures. Thepolynucleotide or polypeptide is included in the composition in anamount and in a form effective to promote stimulation of VEGF receptorsin a blood vessel of the mammalian subject, thereby preventing stenosisor restenosis of the blood vessel.

In a preferred embodiment, the mammalian subject is a human subject. Forexample, the subject is a person suffering from coronary artery diseasethat has been identified by a cardiologist as a candidate who couldbenefit from a therapeutic balloon angioplasty (with or withoutinsertion of an intravascular stent) procedure or from a coronary bypassprocedure. Practice of methods of the invention in other mammaliansubjects, especially mammals that are conventionally used as models fordemonstrating therapeutic efficacy in humans (e.g., primate, porcine,canine, or rabbit animals), also is contemplated.

Polypeptides according to the invention may be administered in anysuitable manner using an appropriate pharmaceutically-acceptablevehicle, e.g., a pharmaceutically-acceptable diluent, adjuvant,excipient or carrier. The composition to be administered according tomethods of the invention preferably comprises (in addition to thepolynucleotide or vector) a pharmaceutically-acceptable carrier solutionsuch as water, saline, phosphate-buffered saline, glucose, or othercarriers conventionally used to deliver therapeutics intravascularly.Multi-gene therapy is also contemplated, in which case the compositionoptionally comprises both the polynucleotide of the invention/vector andanother polynucleotide/vector selected to prevent restenosis. Exemplarycandidate genes/vectors for co-transfection with transgenes encodingpolypeptides of the invention are described in the literature citedabove, including genes encoding cytotoxic factors, cytostatic factors,endothelial growth factors, and smooth muscle cell growth/migrationinhibitors.

The “administering” that is performed according to the present methodmay be performed using any medically-accepted means for introducing atherapeutic directly or indirectly into the vasculature of a mammaliansubject, including but not limited to injections (e.g., intravenous,intramuscular, subcutaneous, or catheter); oral ingestion; intranasal ortopical administration; and the like. In a preferred embodiment,administration of the composition comprising a polynucleotide of theinvention is performed intravascularly, such as by intravenous,intra-arterial, or intracoronary arterial injection. The therapeuticcomposition may be delivered to the patient at multiple sites. Themultiple administrations may be rendered simultaneously or may beadministered over a period of several hours. In certain cases it may bebeneficial to provide a continuous flow of the therapeutic composition.Additional therapy may be administered on a period basis, for example,daily, weekly or monthly.

In general, peroral dosage forms for the therapeutic delivery ofpeptides is ineffective because in order for such a formulation to theefficacious, the peptide must be protected from the enzymaticenvironment of the gastrointestinal tract. Additionally, the peptidemust be formulated such that it is readily absorbed by the epithelialcell barrier in sufficient concentrations to effect a therapeuticoutcome. The peptides of the present invention may be formulated withuptake or absorption enhancers to increase their efficacy. Such enhancerinclude for example, salicylate, glycocholate/linoleate, glycolate,aprotinin, bacitracin, SDS caprate and the like. For an additionaldiscussion of oral formulations of peptides for therapeutic delivery,those of skill in the art are referred to Fix (J. Pharm. Sci., 85(12)1282-1285, 1996) and Oliyai and Stella (Ann. Rev. Pharmacol. Toxicol.,32:521-544, 1993).

The amounts of peptides in a given dosage will vary according to thesize of the individual to whom the therapy is being administered as wellas the characteristics of the disorder being treated. In exemplarytreatments, it may be necessary to administer about 50 mg/day, 75mg/day, 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day. Theseconcentrations may be administered as a single dosage form or asmultiple doses.

The polypeptides may also be employed in accordance with the presentinvention by expression of such polypeptide in vivo, which is oftenreferred to as gene therapy. The present invention provides arecombinant DNA vector containing a heterologous segment encoding apolypeptide of the invention that is capable of being inserted into amicroorganism or eukaryotic cell and that is capable of expressing theencoded protein.

In a highly preferred embodiment, the composition is administeredlocally. Thus, in the context of treating restenosis or stenosis,administration directly to the site of angioplasty or bypass ispreferred. For example, the administering comprises a catheter-mediatedtransfer of the transgene-containing composition into a blood vessel ofthe mammalian subject, especially into a coronary artery of themammalian subject. Exemplary materials and methods for local deliveryare reviewed in Lincoff et al., Circulation, 90: 2070-2084 (1994); andWilensky et al., Trends Cardiovasc. Med., 3:163-170 (1993), bothincorporated herein by reference. For example, the composition isadministered using infusion-perfusion balloon catheters (preferablymicroporous balloon catheters) such as those that have been described inthe literature for intracoronary drug infusions. See, e.g., U.S. Pat.No. 5,713,860 (Intravascular Catheter with Infusion Array); U.S. Pat.No. 5,087,244; U.S. Pat. No. 5,653,689; and Wolinsky et al., J. Am.Coll. Cardiol., 15: 475-481 (1990) (Wolinsky Infusion Catheter); andLambert et al., Coron. Artery Dis., 4: 469-475 (1993), all of which areincorporated herein by reference in their entirety. Use of suchcatheters for site-directed somatic cell gene therapy is described,e.g., in Mazur et al., Texas Heart Institute Journal, 21; 104-111(1994), incorporated herein by reference. In an embodiment where thetransgene encoding a polypeptide of the invention is administered in anadenovirus vector, the vector is preferably administered in apharmaceutically acceptable carrier at a titer of 10⁷-10¹³ viralparticles, and more preferably at a titer of 10⁹-10¹¹ viral particles.The adenoviral vector composition preferably is infused over a period of15 seconds to 30 minutes, more preferably 1 to 10 minutes.

For example, in patients with angina pectoris due to a single ormultiple lesions in coronary arteries and for whom PTCA is prescribed onthe basis of primary coronary angiogram findings, an exemplary protocolinvolves performing PTCA through a 7 F guiding catheter according tostandard clinical practice using the femoral approach. If an optimalresult is not achieved with PTCA alone, then an endovascular stent alsois implanted. (A nonoptimal result is defined as residual stenosisof >30% of the luminal diameter according to a visual estimate, and B orC type dissection.) Arterial gene transfer at the site of balloondilatation is performed with a replication-deficient adenoviral vectorexpressing a polypeptide of the invention immediately after theangioplasty, but before stent implantation, using an infusion-perfusionballoon catheter. The size of the catheter will be selected to match thediameter of the artery as measured from the angiogram, varying, e.g.,from 3.0 to 3.5 F in diameter. The balloon is inflated to the optimalpressure and gene transfer is performed during a 10 minute infusion atthe rate of 0.5 ml/min with virus titer of 1.15×10¹⁰.

In another embodiment, intravascular administration with a gel-coatedcatheter is contemplated, as has been described in the literature tointroduce other transgenes. See, e.g., U.S. Pat. No. 5,674,192 (Cathetercoated with tenaciously-adhered swellable hydrogel polymer); Riessen etal., Human Gene Therapy, 4: 749-758 (1993); and Steg et al.,Circulation, 96: 408-411 (1997) and 90: 1648-1656 (1994); allincorporated herein by reference. Briefly, DNA in solution (e.g., apolynucleotide of the invention) is applied one or more times ex vivo tothe surface of an inflated angioplasty catheter balloon coated with ahydrogel polymer (e.g., Slider with Hydroplus, Mansfield BostonScientific Corp., Watertown, Mass.). The Hydroplus coating is ahydrophilic polyacrylic acid polymer that is cross-linked to the balloonto form a high molecular weight hydrogel tightly adhered to the balloon.The DNA covered hydrogel is permitted to dry before deflating theballoon. Re-inflation of the balloon intravascularly, during anangioplasty procedure, causes the transfer of the DNA to the vesselwall.

In yet another embodiment, an expandable elastic membrane or similarstructure mounted to or integral with a balloon angioplasty catheter orstent is employed to deliver the transgene encoding a polypeptide of theinvention. See, e.g., U.S. Pat. Nos. 5,707,385, 5,697,967, 5,700,286,5,800,507, and 5,776,184, all incorporated by reference herein.

In another variation, the composition containing the transgene encodinga polypeptide of the invention is administered extravascularly, e.g.,using a device to surround or encapsulate a portion of vessel. See,e.g., International Patent Publication WO 98/20027, incorporated hereinby reference, describing a collar that is placed around the outside ofan artery (e.g., during a bypass procedure) to deliver a transgene tothe arterial wall via a plasmid or liposome vector.

In still another variation, endothelial cells or endothelial progenitorcells are transfected ex vivo with the transgene encoding a polypeptideof the invention, and the transfected cells as administered to themammalian subject. Exemplary procedures for seeding a vascular graftwith genetically modified endothelial cells are described in U.S. Pat.No. 5,785,965, incorporated herein by reference.

In preferred embodiments, polynucleotides of the invention furthercomprises additional sequences to facilitate the gene therapy. In oneembodiment, a “naked” transgene encoding a polypeptide of the invention(i.e., a transgene without a viral, liposomal, or other vector tofacilitate transfection) is employed for gene therapy. In thisembodiment, the polynucleotide of the invention preferably comprises asuitable promoter and/or enhancer sequence (e.g., cytomegaloviruspromoter/enhancer [Lehner et al., J. Clin. Microbiol., 29:2494-2502(1991); Boshart et al., Cell, 41:521-530 (1985)]; Rous sarcoma viruspromoter [Davis et al., Hum. Gene Ther., 4:151 (1993)]; Tie promoter[Korhonen et al., Blood, 86(5): 1828-1835 (1995)]; or simian virus 40promoter) for expression in the target mammalian cells, the promoterbeing operatively linked upstream (i.e., 5′) of the polypeptide-codingsequence. The polynucleotides of the invention also preferably furtherincludes a suitable polyadenylation sequence (e.g., the SV40 or humangrowth hormone gene polyadenylation sequence) operably linked downstream(i.e., 3′) of the polypeptide-coding sequence. The polynucleotides ofthe invention also preferably comprise a nucleotide sequence encoding asecretory signal peptide fused in-frame with the polypeptide sequence.The secretory signal peptide directs secretion of the polypeptide of theinvention by the cells that express the polynucleotide, and is cleavedby the cell from the secreted polypeptide. The signal peptide sequencecan be that of another secreted protein, or can be a completelysynthetic signal sequence effective to direct secretion in cells of themammalian subject.

The polynucleotide may further optionally comprise sequences whose onlyintended function is to facilitate large-scale production of the vector,e.g., in bacteria, such as a bacterial origin of replication and asequence encoding a selectable marker. However, in a preferredembodiment, such extraneous sequences are at least partially cleaved offprior to administration to humans according to methods of the invention.One can manufacture and administer such polynucleotides to achievesuccessful gene therapy using procedures that have been described in theliterature for other transgenes. See, e.g., Isner et al., Circulation,91: 2687-2692 (1995); and Isner et al., Human Gene Therapy, 7: 989-1011(1996); incorporated herein by reference in the entirety.

Any suitable vector may be used to introduce the transgene encoding oneof the polypeptides of the invention, into the host. Exemplary vectorsthat have been described in the literature include replication-deficientretroviral vectors, including but not limited to lentivirus vectors [Kimet al., J. Virol., 72(1): 811-816 (1998); Kingsman & Johnson, ScripMagazine, October, 1998, pp. 43-46.]; adeno-associated viral vectors[U.S. Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No.5,622,856; U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat.No. 5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S.Pat. No. 5,851,521; U.S. Pat. No. 5,252,479; Gnatenko et al., J.Investig. Med., 45: 87-98 (1997)]; adenoviral vectors [See, e.g., U.S.Pat. No. 5,792,453; U.S. Pat. No. 5,824,544; U.S. Pat. No. 5,707,618;U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat. No.5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584(1992); Stratford-Perricadet et al., J. Clin. Invest., 90: 626-630(1992); and Rosenfeld et al., Cell, 68: 143-155 (1992)]; anadenoviral-adenoassociated viral hybrid (see for example, U.S. Pat. No.5,856,152) or a vaccinia viral or a herpesviral (see for example, U.S.Pat. No. 5,879,934; U.S. Pat. No. 5,849,571; U.S. Pat. No. 5,830,727;U.S. Pat. No. 5,661,033; U.S. Pat. No. 5,328,688; Lipofectin-mediatedgene transfer (BRL); liposomal vectors [See, e.g., U.S. Pat. No.5,631,237 (Liposomes comprising Sendai virus proteins)]; andcombinations thereof. All of the foregoing documents are incorporatedherein by reference in their entirety. Replication-deficient adenoviralvectors constitute a preferred embodiment.

Other non-viral delivery mechanisms contemplated include calciumphosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467,1973; Chen and Okayama, Mol. Cell. Biol., 7:2745-2752, 1987; Rippe etal., Mol. Cell Biol., 10:689-695, 1990) DEAE-dextran (Gopal, Mol. CellBiol., 5:1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell.Biol., 6:716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA,81:7161-7165, 1984), direct microinjection (Harland and Weintraub, J.Cell Biol., 101:1094-1099, 1985), DNA-loaded liposomes (Nicolau andSene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc.Natl. Acad. Sci. USA, 76:3348-3352, 1979; Felgner, Sci Am. 276(6):102-6,1997; Felgner, Hum Gene Ther. 7(15):1791-3, 1996), cell sonication(Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987),gene bombardment using high velocity microprojectiles (Yang et al.,Proc. Natl. Acad. Sci. USA, 87:9568-9572, 1990), and receptor-mediatedtransfection (Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987; Wu and Wu,Biochemistry, 27:887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev.,12:159-167, 1993).

In a particular embodiment of the invention, the expression construct(or indeed the peptides discussed above) may be entrapped in a liposome.Liposomes are vesicular structures characterized by a phospholipidbilayer membrane and an inner aqueous medium. Multilamellar liposomeshave multiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, In: Liver diseases,targeted diagnosis and therapy using specific receptors and ligands, WuG, Wu C ed., New York: Marcel Dekker, pp. 87-104, 1991). The addition ofDNA to cationic liposomes causes a topological transition from liposomesto optically birefringent liquid-crystalline condensed globules (Radleret al., Science, 275(5301):810-4, 1997). These DNA-lipid complexes arepotential non-viral vectors for use in gene therapy and delivery.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Also contemplated in the presentinvention are various commercial approaches involving “lipofection”technology. In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989).In other embodiments, the liposome may be complexed or employed inconjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Katoet al., J. Biol. Chem., 266:3361-3364, 1991). In yet furtherembodiments, the liposome may be complexed or employed in conjunctionwith both HVJ and HMG-1. In that such expression constructs have beensuccessfully employed in transfer and expression of nucleic acid invitro and in vivo, then they are applicable for the present invention.

Other vector delivery systems that can be employed to deliver a nucleicacid encoding a therapeutic gene into cells include receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993, supra).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987, supra) and transferrin (Wagner et al., Proc. Nat'l. Acad.Sci. USA, 87(9):3410-3414, 1990). Recently, a synthetic neoglycoprotein,which recognizes the same receptor as ASOR, has been used as a genedelivery vehicle (Ferkol et al., FASEB J., 7:1081-1091, 1993; Perales etal., Proc. Natl. Acad. Sci., USA 91:4086-4090, 1994) and epidermalgrowth factor (EGF) has also been used to deliver genes to squamouscarcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (Methods Enzymol., 149:157-176,1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside,incorporated into liposomes and observed an increase in the uptake ofthe insulin gene by hepatocytes. Thus, it is feasible that a nucleicacid encoding a therapeutic gene also may be specifically delivered intoa particular cell type by any number of receptor-ligand systems with orwithout liposomes.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above thatphysically or chemically permeabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al. (Proc. Nat. Acad. Sci.USA, 81:7529-7533, 1984) successfully injected polyomavirus DNA in theform of CaPO4 precipitates into liver and spleen of adult and newbornmice demonstrating active viral replication and acute infection.Benvenisty and Neshif (Proc. Nat. Acad. Sci. USA, 83:9551-9555, 1986)also demonstrated that direct intraperitoneal injection of CaPO₄precipitated plasmids results in expression of the transfected genes.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., Nature, 327:70-73, 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., Proc. Natl. Acad. Sci. USA, 87:9568-9572, 1990). Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten or gold beads.

In embodiments employing a viral vector, preferred polynucleotides stillinclude a suitable promoter and polyadenylation sequence as describedabove. Moreover, it will be readily apparent that, in these embodiments,the polynucleotide further includes vector polynucleotide sequences(e.g., adenoviral polynucleotide sequences) operably connected to thesequence encoding a polypeptide of the invention.

Thus, in one embodiment the composition to be administered comprises avector, wherein the vector comprises a polynucleotide of the invention.In a preferred embodiment, the vector is an adenovirus vector. In ahighly preferred embodiment, the adenovirus vector isreplication-deficient, i.e., it cannot replicate in the mammaliansubject due to deletion of essential viral-replication sequences fromthe adenoviral genome. For example, the inventors contemplate a methodwherein the vector comprises a replication-deficient adenovirus, theadenovirus comprising the polynucleotide of the invention operablyconnected to a promoter and flanked on either end by adenoviralpolynucleotide sequences.

Similarly, the invention includes kits which comprise compounds orcompositions of the invention packaged in a manner which facilitatestheir use to practice methods of the invention. In a simplestembodiment, such a kit includes a compound or composition describedherein as useful for practice of the invention (e.g., polynucleotides orpolypeptides of the invention), packaged in a container such as a sealedbottle or vessel, with a label affixed to the container or included inthe package that describes use of the compound or composition topractice the method of the invention. Preferably, the compound orcomposition is packaged in a unit dosage form. In another embodiment, akit of the invention includes a composition of both a polynucleotide orpolypeptide packaged together with a physical device useful forimplementing methods of the invention, such as a stent, a catheter, anextravascular collar, a polymer film, or the like. In anotherembodiment, a kit of the invention includes compositions of both apolynucleotide or polypeptide of the invention packaged together with ahydrogel polymer, or microparticle polymers, or other carriers describedherein as useful for delivery of the polynucleotides or polypeptides tothe patient.

The polypeptides of the present invention are useful in diagnostic orprognostic assays for detecting VEGF receptor protein expression.Polypeptides of the invention that bind to one or more VEGF receptorsmay be used for detecting and measuring the presence of specificreceptor proteins in samples for purposes such as e.g., medical imaging,detection, screening, or targeted therapy. Detectable labels such asradioactive or non-radioactive labels, including enzyme labels or labelsof the biotin/avidin system, may be used to tag the polypeptide of theinvention. The polypeptide may also be covalently or non-covalentlycoupled to a suitable supermagnetic, paramagnetic, electron dense,ecogenic or radioactive agent for imaging.

The present invention also relates to a diagnostic assay for detectingaltered levels of VEGF receptor proteins in various tissues sinceover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, abnormal cell growth or differentiation.Polypeptides of the invention can be used to quantify future metastaticrisk by assaying biopsy material for the presence of active receptors orligands in a binding assay or kit using detectably-labeled polypeptidesof the invention.

A related aspect of the invention is a method for the detection ofspecific cells, e.g., endothelial cells. These cells may be found invivo, or in ex vivo biological tissue samples. The method of detectioncomprises the steps of contacting a biological tissue comprising, e.g.,endothelial cells, with a hybrid polypeptide according to the inventionwhich is capable of binding to VEGFR(s), under conditions wherein thehybrid polypeptide binds to the cells, optionally washing the biologicaltissue, and detecting the hybrid polypeptide bound to the cells in thebiological tissue, thereby detecting the cells. It will be apparent thatcertain polypeptides of the invention are useful for detecting and/orimaging cells that express more than one VEGFR, whereas otherpolypeptides are useful for imaging cells which specifically express aparticular VEGFR.

The invention also is directed to a method for imaging vertebrate tissuesuspected of containing cells that express a specific VEGFR comprisingthe steps of: (a) contacting vertebrate tissue with a compositioncomprising polypeptide(s) of the invention that specifically bind theparticular VEGFR; and (b) imaging the tissue by detecting theVEGFR-binding polypeptide bound to the tissue. Preferably, the tissue ishuman tissue, and the method further comprises the step of washing thetissue, after the contacting step and before the imaging step, underconditions that remove from the tissue polypeptides that are not boundto the VEGFR in the tissue.

In a related variation, the invention provides a method for imagingtumors in tissue from a vertebrate organism, comprising the steps of:(a) contacting vertebrate tissue suspected of containing a tumor with acomposition comprising a VEGFR binding compound; (b) detecting the VEGFRbinding compound bound to cells in said tissue; and (c) imaging solidtumors by identifying blood vessel endothelial cells bound by the VEGFRbinding compound, wherein blood vessels expressing VEGFR are correlatedwith the presence and location of a tumor in the tissue.

The present invention also is directed to the use of hybrid polypeptidesof the invention that bind VEGF receptors as specific markers forparticular tissues and cell types. For example, those polypeptides ofthe invention that specifically bind VEGFR-3 can serve as markers forlymphatic endothelial cells.

Similarly, polypeptides of the invention may be screened for an abilityto modulate the growth of isolated cells or cell lines. For example,certain neoplastic disease states are characterized by the appearance ofVEGF receptors on cell surfaces [Valtola et al., Am J Path 154:1381-90(1999)]. Polypeptides of the invention may be screened to determine theability of the polypeptide to modulate the growth of the neoplasticcells. Other disease states are likely characterized by mutations inVEGF receptors [Ferrell et al., Hum Mol Genetics 7:2073-78 (1998)].Polypeptides of the invention that modulate the activity of the mutantforms of the VEGF receptor in a manner different thannaturally-occurring vascular endothelial growth factors will be usefulat modulating the symptoms and severity of the such disease states.

In vivo imaging or tissue biopsy may reveal that certain neoplasticcells are expressing a particular combination of receptors, therebyproviding an indication for polypeptides of the invention that bind theexpressed set of receptors and inhibit ligand mediated growth.

The use of such diagnostic imaging is particularly suitable in obtainingan image of, for example, a tumor mass or the neovascularization near atumor mass. It is contemplated that the peptides of the presentinvention may be employed for imaging in a manner analogous to theantibody-based methods disclosed in U.S. Pat. No. 6,107,046,incorporated herein by reference.

Many appropriate imaging agents are known in the art, as are methods ofattaching the labeling agents to the peptides of the invention (see,e.g., U.S. Pat. No. 4,965,392, U.S. Pat. No. 4,472,509, U.S. Pat. No.5,021,236 and U.S. Pat. No. 5,037,630, incorporated herein byreference). The labeled peptides are administered to a subject in apharmaceutically acceptable carrier, and allowed to accumulate at atarget site having the VEGFR-3 receptor. This peptide imaging agent thenserves as a contrast reagent for X-ray, magnetic resonance, sonographicor scintigraphic imaging of the target site. The peptides of the presentinvention are a convenient and important addition to the availablearsenal of medical imaging tools for the diagnostic investigation ofcancer and other VEGFR-3 related disorders.

Paramagnetic ions useful in the imaging agents of the present inventioninclude for example chromium (III), manganese (II), iron (III), iron(II), cobalt (II), nickel (II) copper (II), neodymium (III), samarium(III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III). Ions useful for X-rayimaging include but are not limited to lantanum (III), gold(III), lead(II) and particularly bismuth (III). Radioisotopes for diagnosticapplications include for example, ²¹¹astatine, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁶⁷copper, ¹⁵²Eu, ⁶⁷gallium, ³hydrogen, ²³iodine,¹²⁵iodine, ¹¹¹indium, ⁵⁹iron, ³²phosphorus, ¹⁸⁶rhenium, ⁷⁵selenium,³⁵sulphur, ^(99m)technicium and ⁹⁰yttrium.

The peptides of the present invention may be labeled according totechniques well known to those of skill in the art. For example, thepeptides can be iodinated by contacting the peptide with sodium orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite or an enzymatic oxidant such as lactoperoxidase. Peptidesmay be labeled with technetium-99m by ligand exchange, for example, byreducing pertechnate with stannous solution, chelating the reducedtechnetium onto a Sephadex column and applying the peptide to thecolumn. These and other techniques for labeling proteins and peptidesare well known to those of skill in the art.

Using Polypeptides of the Invention in Combined Therapy for NeoplasticDisorders

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. As describedabove, the peptides of the present invention may be administered inconjunction with chemo- or radiotherapeutic intervention, immunotherapy,or with other anti-angiogenic/anti-lymphangiogenic therapy.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells via combination therapy, using the methods and compositionsof the present invention, one would generally contact a “target” cell ortissue, (e.g., a tumor and/or its vasculature) with the therapeuticpeptides of the present invention (either as a peptide composition or asan expression construct that will express the peptide) and at least oneother agent, which optionally is conjugated to the peptide of theinvention. These compositions would be provided in a combined amounteffective to kill or inhibit proliferation of the cancer by killingand/or inhibiting the proliferation of the cancer cells and/or theendothelia of blood and lymphatic vessels supplying and serving thecancer cells. This process may involve contacting the cells with thepeptide or expression construct and the agent(s) or factor(s) at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the peptide orexpression construct and the other includes the second agent.

Alternatively, the therapeutic treatment employing the peptides of thepresent invention may precede or follow the other agent treatment byintervals ranging from minutes to weeks. In embodiments where the otheragent and expression construct are administered separately, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and thepeptide-based therapeutic would still be able to exert an advantageouslycombined effect. In such instances, it is contemplated that one wouldadminister both modalities within about 12-24 hours of each other and,more preferably, within about 6-12 hours of each other, with a delaytime of only about 12 hours being most preferred. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.Repeated treatments with one or both agents is specificallycontemplated. In specific embodiments, an anti-cancer therapy may bedelivered which directly attacks the cancer cells in a manner to kill,inhibit or necrotize the cancer cell, in addition a therapeuticcomposition based on the peptides of the present invention also isadministered to the individual in amount effective to have anantiangiogenic and/or anti-lymphangiogenic effect. The peptidecompositions may be administered following the other anti-cancer agent,before the other anti-cancer agent or indeed at the same time as theother anti-cancer agent, optionally conjugated to the other agent.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as chemotherapeutic agents,” function toinduce DNA damage, all of which are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated to be of use, include, e.g., adriamycin, 5-fluorouracil(5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. The invention alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide.

In treating cancer according to the invention, one would contact thetumor cells and/or the endothelia of the tumor vessels with an agent inaddition to the therapeutic agent comprising one or more peptide of thepresent invention. This may be achieved by irradiating the localizedtumor site with radiation such as X-rays, UV-light, gamma-rays or evenmicrowaves. Alternatively, the tumor cells may be contacted with theagent by administering to the subject a therapeutically effective amountof a pharmaceutical composition comprising a compound such as,adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D,mitomycin C, or cisplatin. Kinase inhibitors also contemplated to beuseful in combination therapies with the peptides of the presentinvention. The agent may be prepared and used as a combined therapeuticcomposition, or kit, by combining it with a chimeric peptide of theinvention, as described above.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with chimeric peptide-based therapy. Agentssuch as cisplatin, and other DNA alkylating agents may be used.Cisplatin has been widely used to treat cancer, with efficacious dosesused in clinical applications of 20 mg/m² for 5 days every three weeksfor a total of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

By way of example the following is a list of chemotherapeutic agents andthe cancers which have been shown to be managed by administration ofsuch agents. Combinations of these chemotherapeutics with the peptidesof the present invention may prove to be useful in amelioration ofvarious neoplastic disorders. Examples of these compounds includeadriamycin (also known as doxorubicin), VP-16 (also known as etoposide),and the like, daunorubicin (intercalates into DNA, blocks DNA-directedRNA polymerase and inhibits DNA synthesis); mitomycin (also known asmutamycin and/or mitomycin-C) is an antibiotic isolated from the brothof Streptomyces caespitosus which has been shown to have antitumoractivity; Actinomycin D also may be a useful drug to employ incombination with the peptides of the present invention because tumorswhich fail to respond to systemic treatment sometimes respond to localperfusion with dactinomycin which also is known to potentiateradiotherapy. It also is used in combination with primary surgery,radiotherapy, and other drugs, particularly vincristine andcyclophosphamide and has been found to be effective against Ewing'stumor, Kaposi's sarcoma, and soft-tissue sarcomas, choriocarcinoma,metastatic testicular carcinomas, Hodgkin's disease and non-Hodgkin'slymphomas.

Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolatedfrom a strain of Streptomyces verticillus, is effective in themanagement of the following neoplasms either as a single agent or inproven combinations with other approved chemotherapeutic agents insquamous cell carcinoma such as head and neck (including mouth, tongue,tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa,gingiva, epiglottis, larynx), skin, penis, cervix, and vulva. It hasalso been used in the treatment of lymphomas and testicular carcinoma.

Cisplatin has been widely used to treat cancers such as metastatictesticular or ovarian carcinoma, advanced bladder cancer, head or neckcancer, cervical cancer, lung cancer or other tumors and may be a usefulcombination with the peptides of the present invention. VP16 (etoposide)and is used primarily for treatment of testicular tumors, in combinationwith bleomycin and cisplatin, and in combination with cisplatin forsmall-cell carcinoma of the lung. It is also active againstnon-Hodgkin's lymphomas, acute nonlymphocytic leukemia, carcinoma of thebreast, and Kaposi's sarcoma associated with acquired immunodeficiencysyndrome (AIDS). Tumor Necrosis Factor [TNF; Cachectin] glycoproteinthat kills some kinds of cancer cells, activates cytokine production,activates macrophages and endothelial cells, promotes the production ofcollagen and collagenases, is an inflammatory mediator and also amediator of septic shock, and promotes catabolism, fever and sleep. TNFcan be quite toxic when used alone in effective doses, so that theoptimal regimens probably will use it in lower doses in combination withother drugs. Its immunosuppressive actions are potentiated byγ-interferon, so that the combination potentially is dangerous. A hybridof TNF and interferon-α also has been found to possess anti-canceractivity.

Taxol an antimitotic agent original isolated from the bark of the ashtree, Taxus brevifolia, and its derivative paclitaxol have proven usefulagainst breast cancer and may be used in the combination therapies ofthe present invention. Beneficial responses to vincristine have beenreported in patients with a variety of other neoplasms, particularlyWilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, andcarcinomas of the breast, bladder, and the male and female reproductivesystems. Vinblastine also is indicated as a useful therapeutic in thesame cancers as vincristine. The most frequent clinical use ofvinblastine is with bleomycin and cisplatin in the curative therapy ofmetastatic testicular tumors. It is also active in Kaposi's sarcoma,neuroblastoma, and Letterer-Siwe disease (histiocytosis X), as well asin carcinoma of the breast and choriocarcinoma in women.

Melphalan also known as alkeran, L-phenylalanine mustard, phenylalaninemustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative ofnitrogen mustard. Melphalan is a bifunctional alkylating agent which isactive against selective human neoplastic diseases. Melphalan is theactive L-isomer of the D-isomer, known as medphalan, which is lessactive against certain animal tumors, and the dose needed to produceeffects on chromosomes is larger than that required with the L-isomer.Melphalan is available in form suitable for oral administration and hasbeen used to treat multiple myeloma. Available evidence suggests thatabout one third to one half of the patients with multiple myeloma show afavorable response to oral administration of the drug. Melphalan hasbeen used in the treatment of epithelial ovarian carcinoma.

Cyclophosphamide is stable in the gastrointestinal tract, tolerated welland effective by the oral and parental routes and does not cause localvesication, necrosis, phlebitis or even pain. Chlorambucil, abifunctional alkylating agent of the nitrogen mustard type that has beenfound active against selected human neoplastic diseases. Chlorambucil isindicated in the treatment of chronic lymphatic (lymphocytic) leukemia,malignant lymphomas including lymphosarcoma, giant follicular lymphomaand Hodgkin's disease. It is not curative in any of these disorders butmay produce clinically useful palliation.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as gamma-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors effect abroad range of damage DNA, on the precursors of DNA, the replication andrepair of DNA, and the assembly and maintenance of chromosomes. Dosageranges for X-rays range from daily doses of 50 to 200 roentgens forprolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells. (See, e.g., Remington'sPharmaceutical Sciences” 15th Edition, chapter 33, in particular pages624-652.) Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

In addition to combining chimeric peptide-based therapies with chemo-and radiotherapies, it also is contemplated that combination with genetherapies will be advantageous. For example, targeting of chimericpeptide-based therapies and p53 or p16 mutations at the same time mayproduce an improved anti-cancer treatment. Any other tumor-related geneconceivably can be targeted in this manner, for example, p21, Rb, APC,DCC, NF-1, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC,MCC, ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl andabl.

In addition to the anticancer therapeutics discussed above, it iscontemplated that the peptides of the invention may be combined withother angiogenesis inhibitors. The peptides of the present invention areexpected to have both anti-lymphangiogenic and anti-angiogenicproperties. Many anti-angiogenic drugs also may haveanti-lymphangiogenic properties.http://cancertrials.nci.nih.gov/news/angio is a website maintained bythe National Institutes of Health which provides current information onthe trials presently being conducted with anti-angiogenic agents. Theseagents include, for example, Marimastat (British Biotech, Annapolis Md.;indicated for non-small cell lung, small cell lung and breast cancers);AG3340 (Agouron, La Jolla, Calif.; for glioblastoma multiforme); COL-3(Collagenex, Newtown Pa.; for brain tumors); Neovastat (Aeterna, Quebec,Canada; for kidney and non-small cell lung cancer) BMS-275291(Bristol-Myers Squibb, Wallingford Conn.; for metastatic non-small cellling cancer); Thalidomide (Celgen; for melanoma, head and neck cancer,ovarian, metastatic prostate, and Kaposi's sarcoma; recurrent ormetastatic colorectal cancer (with adjuvants); gynecologic sarcomas,liver cancer; multiple myeloma; CLL, recurrent or progressive braincancer, multiple myeloma, non-small cell lung, nonmetastatic prostate,refractory multiple myeloma, and renal cancer); Squalamine (MagaininPharmaceuticals Plymouth Meeting, Pa.; non-small cell cancer and ovariancancer); Endostatin (EntreMEd, Rockville, Md.; for solid tumors); SU5416(Sugen, San Francisco, Calif.; recurrent head and neck, advanced solidtumors, stage IIIB or IV breast cancer; recurrent or progressive brain(pediatric); Ovarian, AML; glioma, advanced malignancies, advancedcolorectal, von-Hippel Lindau disease, advanced soft tissue; prostatecancer, colorectal cancer, metastatic melanoma, multiple myeloma,malignant mesothelioma: metastatic renal, advanced or recurrent head andneck, metastatic colorectal cancer); SU6668 (Sugen San Francisco,Calif.; advanced tumors); interferon-α; Anti-VEGF antibody (NAtionalCancer Institute, Bethesda Md.; Genentech San Francisco, Calif.;refractory solid tumors; metastatic renal cell cancer, in untreatedadvanced colorectal); EMD121974 (Merck KCgaA, Darmstadt, Germany; HIVrelated Kaposi's Sarcoma, progressive or recurrent Anaplastic Glioma);Interleukin 12 (Genetics Institute, Cambridge, Mass.; Kaposi's sarcoma)and IM862 (Cytran, Kirkland, Wash.; ovarian cancer, untreated metastaticcancers of colon and rectal origin and Kaposi's sarcoma). Theparenthetical information following the agents indicates the cancersagainst which the agents are being used in these trials. It iscontemplated that any of these disorders may be treated with thepeptides of the present invention either alone or in combination withthe agents listed.

Additional features of the invention will be apparent from the followingExamples.

EXAMPLE 1 Construction of VEGF-A/VEGF-C Hybrid Molecules

Although the amino acid residues of the receptor binding domain of VEGFfamily members are share conserved motifs, these proteins exhibitdifferent receptor specificities. In the following experiment, DNAmolecules encoding polypeptides containing different portions of thereceptor binding domains of either VEGF-A or VEGF-C were constructedusing a combinatorial approach to create novel hybrid molecules withunique structural and functional characteristics.

To generate the novel molecules, the nucleotide sequences of VEGF-A andmature VEGF-C were analyzed to determine localized regions of nucleotideidentity which would be suitable for designing short DNA fragments whichcould be synthesized and readily recombined. Eight corresponding regionsof identity were selected in each molecule as sites for fragmentization(into nine fragments) and recombination into chimeric (hybrid)molecules. These fragmentation sites were chosen based on nucleotidesequence and also because the resultant fragments would correspond tostructural elements (e.g., alpha helix, loop, etc.) based on the crystalstructure of VEGF-A (see FIG. 1).

Fragmentation of VEGF-A

Nine pairs of synthetic oligonucleotides were designed based on thecoding sequence for VEGF₁₂₁, for the purpose of forming nine DNAfragments that encompass the receptor binding domain encoding region ofVEGF-A (corresponding to nucleotides 156 to 461 of SEQ ID NO: 1, whichencode amino acid residues 34 to 135 of SEQ ID NO: 2). Eacholigonucleotide pair comprised a forward primer containing codingsequence and a reverse primer with nucleotide sequence complementary toa portion of the forward primer, to permit annealing of the primers toeach other into a double-stranded DNA fragment. Either the forward orreverse primer of each pair also included a short 5′ and 3′ nucleotidesequence that was not complementary to any sequence of its pairedprimer. These short additional sequences correspond to the localizedregions of nucleotide identity set forth above. Following annealing ofprimer pairs, this additional sequence formed single-stranded overhangscompatible with annealing with other double-stranded annealed primerpairs, as described in greater detail below. The nucleotide sequencesfrom the VEGF-A forward and reverse primers are set forth below inTables 1A and 1B, respectively. TABLE 1A Forward (Coding) Primers forVEGF-A A1-F gat cCT GGG CAG AAT CAT CAC GAA GTG Gtg aaa t D   P   G   Q   N   H   H   E   V   V   K A2-F TC ATG GAT GTC TAT CAGCGC AGC TAC TGC CAT F   M   D   V   Y   Q   R   S   Y   C   H A3-F ccgaTC GAG ACA CTG GTG GAC ATC TTC CAG GAATAGAAGAGC P   I   E   T   L   V   D   I   F   Q A4-F CGCTCTTCGAA TAC CCT GAT GAGATC GAG TAC A          E   Y   P   D   E   I   E   Y A5-F tc ttc aag ccaTCC TGC GTG CCC CTG ATG AGA TGT GGCI   F   K   P   S   C   V   P   L   M   R   C   G A6-F CCG GGT TGC TGCAAT GAC GAA GGG CTG G      G   C   C   N   D   E   G   L A7-F ag tgC GTTCCC ACC GAG GAG TCC AAC ATC ACC ATG CAG ATT ATG AGE   C   V   P   T   E   E   S   N   I   T   M   Q   I   M   R A8-F a attAAA CCT CAC CAA GGG CAG CAC ATC GGA GAG ATG agc ttt   I   K   P   H   Q   G   Q   H   I   G   E   M   S   F A9-F CTC CAGCAT AAC AAA TGT GAA TGT AGA CCA AAG AAA GATTGAGTCTTCGC L   Q   H   N   K   C   E   C   R   P   K   K   D

The nucleotide sequences of forward primers A1-F to A9-F are set forthin SEQ ID NOs: 3-11, respectively. For each of the primers listed, thetop strand shows the DNA sequence and the bottom strand indicates theamino acids encoded by that particular primer, SEQ ID NOs: 128-136,respectively. In some instances, only two nucleotides of a given codonis contained in one primer, and the remaining nucleotide of the codon iscontained in the preceding or following primer. In these cases, theamino acid is listed under the primer that contains 2 out of the 3nucleotides of that particular codon. Boldface type indicate nucleotidescoding for amino acids that constitute a protein linker region and arenot part of the parent VEGF-A or VEGF-C molecule; underlined nucleotidesare those that are removed during assembly of the fragments into hybridconstructs; and the lowercase letters are those nucleotides that producean overhang when the oligonucleotide pairs are annealed to each other toproduce the 9 fragments. TABLE 1B Reverse (Non-Coding) Primers forVEGF-A A1-R CCACTTCGTGATGATTCTGCCCAG A2-RtCggATGGCAGTAGCTGCGCTGATAGACATCCATGAatttca A3-RtcgaGCTCTTCTATTCCTGGAAGATGTCCACCAGTGTCTCGA A4-RtggcttgaagatGTACTCGATCTCATCAGGGTATTCGAAGAGCGg tac A5-RcatgGCCACATCTCATCAGGGGCACGCAGGA A6-RgcactCCAGCCCTTCGTCATTGCAGCAACCCGGGTAC A7-RaattCTCATAATCTGCATGGTGATGTTGGACTCCTCGGTGGGAAC A8-RCATCTCTCCGATGTGCTGCCCTTGGTGAGGTTT A9-RGGCCGCGAAGACTCAATCTTTCTTTGGTCTACATTCACATTTGTT ATGCTGGAGaaagctThe nucleotide sequences of reverse primers A1-R to A9-R are set forthin SEQ ID NOs: 12-20, respectively. Boldface, underlined and lowercaseletters are used as described in Table 1A.

Nine VEGF-A polynucleotide fragments were assembled by annealing amatched pair of synthetic oligonucleotide primers. For example, fragmentA1 was created by annealing primer A1-F with primer A1-R, fragment A2was created by annealing A2-F with A2-R, and so on. Annealing wasaccomplished by incubating 2 pmol/μl of each appropriate primer, 20 mMTris/HCl, 2 mM MgCl₂, and 50 mM NaCl, pH 7.4 at 95° C. for 5 minutes,followed by cooling of the solution to 37° C. at a rate of 1° C./minute.As shown in Table 1A, fragment A1 encodes amino acid residues 34 to 42,and part of amino acid 43 of SEQ ID NO: 2; fragment A2 encodes part ofamino acid 43, and amino acids 44-53 of SEQ ID NO: 2; fragment A3encodes amino acids 54 to 63, and part of amino acid 64 of SEQ ID NO: 2;fragment A4 encodes part of amino acid 64, amino acids 65 to 71, andpart of amino acid 72 of SEQ ID NO: 2; fragment A5 encodes part of aminoacid 72, amino acids 73 to 83, and part of amino acid 84 of SEQ ID NO:2; fragment A6 encodes part of amino acid 84, amino acids 85 to 92, andpart of amino acid 93 of SEQ ID NO: 2; fragment A7 encodes part of aminoacid 93, amino acids 94 to 107, and part of amino acid 108 of SEQ ID NO:2; fragment A8 encodes part of amino acid 108, and amino acids 109 to122 of SEQ ID NO: 2; and fragment A9 encodes amino acids 123 to 135 ofSEQ ID NO: 2.

Fragmentation of VEGF-C

In a similar manner, nine pairs of oligonucleotides were designed andsynthesized based upon the amino acid sequence of the receptor bindingdomain of VEGF-C (corresponding to nucleotides 658 to 999 of SEQ ID NO:21, which encode amino acid residues 112 to 216 of SEQ ID NO: 22). Thenucleotide sequences of the nine forward primers and nine reverseprimers are set forth in Table 2A (SEQ ID NOs: 23-31) and Table 2B (SEQID NOs: 32-40), respectively. The amino acid sequences encoded by thenine forward primers are set forth in SEQ ID NOs: 137-145, respectively.TABLE 2A Forward (Coding) Primers for VEGF-C C1-F gat cCT GCA CAT TATAAT ACC GAG ATC Ctg aaa t  D   P   A   H   Y   N   T   E   I   L   KC2-F CT ATT GAT AAT GAG TGG AGA AAG ACT CAG TGC ATGS   I   D   N   E   W   R   K   T   Q   C   M C3-F ccg aGA GAG GTG TGTATC GAC GTG GGG AAG GAATAGAAGAGC  P   R   E   V   C   I   D   V   G   KC4-F CGCTCTTCGAA TTT GGA GTC GCG ACA AAC ACC T         E   F   G   V   A   T   N   T C5-F tc ttc aag cca CCA TGT GTGTCC GTG TAC AGA TGT GGCF   F   K   P   P   C   V   S   V   Y   R   C   G C6-F CCG GGT TGC TGCAAT AGT GAG GGG CTG C      G   C   C   N   S   E   G   L C7-F ag tgc ATGAAC ACG TCC ACG AGC TAC CTC AGC AAG ACG CTG TTT GAQ   C   M   N   T   S   T   S   Y   L   S   K   T   L   F   E C8-F a attACA GTG CCT CTC TCT CAA GGG CCC AAA CCA GTG ACA ATC agcttt   I   T   V   P   L   S   Q   G   P   K   P   V   T   I   S  F C9-F GCCAAT CAC ACT TCC TGC CGA TGC ATG TCT AAG CTG GATTGAGTCTTCGC A   N   H   T   S   C   R   C   M   S   K   L   D

TABLE 2B Forward (Coding) Primers for VEGF-C C1-RGGATCTCGGTATTATAATGTGCAG C2-RtcggCATGCACTGAGTCTTTCTCCACTCATTATCAATAGatttca C3-RtcgaGCTCTTCTATTCCTTCCCCACGTCGATACACACCTCTC C4-RtggcttgaagaAGGTGTTTGTCGCGACTCCAAATTCGAAGAGCGg tac C5-RcatgGCCACATCTGTACACGGACACACATGG C6-RgcactGCAGCCCCTCACTATTGCAGCAACCCGGgtac C7-RaattTCAAACAGCGTCTTGCTGAGGTAGCTCGTGGACGTGTTCAT C8-RGATTGTCACTGGTTTGGGCCCTTGAGAGAGAGGCACTGT C9-RggccGCGAAGACTCAATCCAGCTTAGACATGCATCGGCAGGAAGT GTGATTGGCaaagct

Boldface, underlined and lowercase letters are used in Tables 2A and 2Bas described in Table 1A.

Primer pairs were annealed to form nine double-stranded DNA fragmentswhich together encoded the receptor binding domain of VEGF-C, and whichpossessed appropriate single stranded overhangs for annealing to otherfragments, as described above for VEGF-A.

Fragment C1 encodes amino acid residues 112 to 120, and part of aminoacid 121 of SEQ ID NO: 22; fragment C2 encodes part of amino acid 121and amino acids 122 to 132 of SEQ ID NO: 22; fragment C3 encodes aminoacids 133 to 142, and part of amino acid 143 of SEQ ID NO: 22; fragmentC4 encodes part of amino acid 143, amino acids 144 to 150, and part ofamino acid 151 of SEQ ID NO: 22; fragment C5 encodes part of amino acid151, amino acids 152 to 162, and part of amino acid 163 of SEQ ID NO:22; fragment C6 encodes part of amino acid 163, and amino acids 164 to171, and part of amino acid 172 of SEQ ID NO: 22; fragment C7 encodespart of amino acid 172, amino acids 173 to 186, and part of amino acid187 of SEQ ID NO: 22; fragment C8 encodes part of amino acid 187, aminoacid 188 to 203 of SEQ ID NO: 22; and fragment C9 encodes amino acid 204to 216 of SEQ ID NO: 22.

Discussion Regarding the Synthesis of the VEGF-A and VEGF-C Fragments

Thus, by synthesizing and annealing nine pairs of primers designed fromthe VEGF-A amino acid sequence and nine pairs of primers designed fromthe VEGF-C amino acid sequence, eighteen DNA fragments were generated.FIG. 2 is a schematic diagram illustrating the construction of the 9VEGF-A and 9 VEGF-C DNA fragments. The oligonucleotides were designed toproduce double-stranded DNA fragments containing unique cohesive endsupon annealing. Ligation of the 9 VEGF-A DNA fragments produces a singlelinear double-stranded DNA encoding amino acids 34-135 of VEGF-A (SEQ IDNO: 2), and ligation of the 9 VEGF-C DNA fragments results in a singleDNA encoding amino acids 112-216 of VEGF-C (SEQ ID NO: 22).

While the insertion of cohesive ends greatly facilitated ligation offragments in a desired order and orientation, it will be appreciatedthat ligation of fragments can also be accomplished without cohesiveends. Blunt-end fragments also can be synthesized and annealed togenerate hybrid proteins using the method described above. With ablunt-end strategy, the nucleotide sequences of the parent molecules donot need to be examined for the presence of nucleotide identity toenable the creation of cohesive ends. However, additional post-ligationscreening may be required to identify hybrids that contain fragments inthe desired order and orientation.

Several additional details regarding the synthetic primers and thedouble-stranded DNA fragments deserve emphasis. First, it is worthnoting that, for VEGF-A fragment A1 and VEGF-C fragment C1, the firsttwo encoded amino acids, Asp and Pro, constitute a protein linker(encoded by an engineered BamHI recognition site) and do not correspondto either VEGF-A or VEGF-C sequences.

Second, referring to FIGS. 1 and 2, it is noteworthy that many of thefragments were designed to correspond to discrete structural elementswithin the receptor binding domain of VEGF family proteins. Fragment 2corresponds to the N-terminal helix; fragment 4 corresponds to β2;fragment 6 corresponds to the β3-β4 loop, fragment 7 corresponds to β5;fragment 8 corresponds to the β5-β6 loop; and fragment 9 corresponds toβ7.

Third, it is noteworthy that the thirty-six oligonucleotides that weredesigned do not correspond exactly with native human VEGF-A or VEGF-CcDNA sequences (i.e., DNA counterparts of naturally-occurring human mRNAsequences), notwithstanding the fact that the oligonucleotides weredesigned to retain encoded amino acid sequences of the human VEGF-A andVEGF-C polypeptides. For example, the oligonucleotides were designedsuch that the native (endogenous) human nucleotide sequence encoding thereceptor binding domain for both VEGF-A and VEGF-C were modified togenerate new restriction sites, to provide longer stretches ofnucleotide identity where overlaps were desired between the “A” and “C”fragments, or to improve codon usage for expression in human cellculture. All nucleotide mutations (relevant to the native sequences)were silent. Thus, the amino acid sequences of the receptor bindingdomain of VEGF-A (resulting from annealing fragments A1-A9) and VEGF-C(from annealing fragments C1-C9) are identical to that of the respectiveparent molecule.

Fourth, referring again to FIG. 2, it is noteworthy that each of thenine VEGF-A fragments aligns with the corresponding VEGF-C fragment, andhas a compatible cohesive end to anneal to adjacent fragments from theother molecule. For example, fragments A1 and C1 correspond to the samerelative portions of VEGF-A and VEGF-C, respectively, and have identicaltop strand cohesive ends. These cohesive ends are exactly complementaryto bottom strand cohesive ends of both fragments A2 and C2, such that A1could anneal to either A2 or C2, and C1 also could anneal to A2 or C2.Fragments A2 and C2 correspond to the same relative portions of VEGF-Aand VEGF-C, and each possesses another bottom strand cohesive end thatis exactly complementary to top strand cohesive ends of fragments A3 andC3, and so on. Thus, each set of nine fragments was designed not only toanneal to adjacent fragments of its parent VEGF-A/VEGF-C molecule, butalso to anneal to adjacent fragments of the other molecule.

Assembly of Chimeric (Hybrid) VEGF Molecules

Assembly of the 9 VEGF-A and 9 VEGF-C DNA fragments into hybrid DNAscontaining regions from both VEGF-A and VEGF-C was accomplished byligating different combinations of the VEGF-A and VEGF-C DNA fragments.All DNA fragments were isolated after digestion with appropriaterestriction enzymes and gel electrophoresis using Qiaex II beads(Qiagen). It will be apparent that, if the proper order(1-2-3-4-5-6-7-8-9) of fragments is preserved, the nine VEGF-A fragmentsand the nine VEGF-C fragments can be recombined and annealed into 512distinct hybrids, two of which represent naturally-occurring sequences(A1-A2-A3-A4-A5-A6-A7-A8-A9 and C1-C2-C3-C4-C5-C6-C7-C8-C9) and 510 ofwhich represent novel hybrids. All 512 sequences were reconstructedusing the following three step process.

First, the receptor binding domains of VEGF-A and VEGF-C were dividedinto 4 subdomains designated N123, N45, C67 and C89, as shown in FIG. 2.N123 consists of the first 3 DNA fragments encoding the receptor bindingdomain of both VEGF-A and VEGF-C. N45, C67 and C89 each consists of 2DNA fragments where N45 includes fragments 4 and 5, C67 consists offragments 6 and 7, and C89 includes fragments 8 and 9.

Continuous DNA's corresponding to the N123 region were constructed byligating fragments 1, 2, and 3 from either VEGF-A or VEGF-C, thusproducing a total of eight possible different N123 DNA segments shownschematically in FIG. 3. Similarly, continuous DNAs corresponding to theN45, C67, and C89 regions were constructed by ligating the twoappropriate DNA fragments from VEGF-A or VEGF-C. In these cases, allfour possible different molecules were produced for each of the regions.FIG. 4 is a schematic diagram illustrating all four possible N45 DNAsegments, FIG. 5 depicts all four possible C67 DNA segments, and FIG. 6shows all four possible C89 DNA segments. All of these molecules werecloned into the multiple cloning site of the pKO-Scrambler-V912-BXvector (Lexicon Genetics Inc.) as part of the ligation reaction. Allligations were carried out by combining 8 nmol/μl of vector cut with theappropriate restriction enzyme that enables cloning of the inserts intothe vector, and dephosphorylated; 80 nmol/μl each of DNA fragments thatare to be inserted into the vector; and 5 Weiss Units of T4 DNA ligasein 50 mM Tris/HCl, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA, and5% PEG-4000, pH 7.5, followed by incubation for 12 hours at 16° C. FIGS.7A-7D depict the amino acid sequences encoded by each of fragments A1-A9and C1-C9; and schematically depict all the permutations of encodedpeptides that result from recombinations that form the eight N123constructs (FIG. 7A), four N45 constructs (FIG. 7B), four C67 constructs(FIG. 7C), and four C89 constructs (FIG. 7D).

In the second step, the N123 fragments were joined with N45 fragments,and the C67 fragments were joined with C89 fragments. The N123 and N45fragments were removed from their pKO-Scrambler-V912 host vector bydigestion with restriction enzymes that allowed ligation of N123 to N45,and which also achieved removal of the non-protein coding regions offragments 3 and 4 (see Tables 1A, 1B, 2A and 2B). By ligating each ofthe eight different N123 regions to all four possible N45 regions, 32distinct N-terminal portions of the receptor binding domains wereobtained. These clones were further inserted into the pSecTagI vector(SEQ ID NO: 41). The pSecTagI vector is a combined E. coli/mammalianexpression vector which was constructed by modifying the pSecTagA vector(Invitrogen). pSecTagA was modified to eliminate specific restrictionsites using site-directed mutagenesis and synthetic linkers, and the EM7promoters from pICZα-A (Invitrogen) and pTRACER-CMV were addeddownstream to the CMV promoter of pSecTagA. Both pSecTagI and it'sparent vector, pSecTagA, allow high level of expression in mammaliancell culture using suitable cell lines e.g., 293T cells, zeocinselection of stably transfected mammalian cells, contain a mammaliansignal peptide for secretion of the expressed protein, and contain aC-terminal myc epitope and polyhistidine tag for detection, quantitationand purification of the expressed protein. The pSecTagI vector differsfrom the pSecTagA vector in that expression in E. coli is constitutiveand modification of the restriction sites facilitated cloning of thehybrid constructs.

The C67 and C89 fragments were removed from their pKO-Scrambler-V912host vector by digestion with appropriate restriction enzymes, whichalso achieved removal of the non-protein coding regions of fragments 6and 9 (see Tables 1A, 1B, 2A and 2B). Ligation of the four different C67molecules to the four different C89 molecules produced 16 distinctC-terminal halves of the receptor binding domain. The C C67-C89fragments were cloned into the pKO-Scrambler vector during theseligations. Finally, 512 final ligations that combined the 32 differentN-terminal portions and the 16 distinct C-terminal regions resulted in atotal of 512 distinct molecules of which 510 are hybrids composed ofboth VEGF-A and VEGF-C amino acid residues. During this step the 512constructs were cloned into the pSecTagI vector which contained the 32different N-terminal portions. The remaining 2 molecules correspond tothe original VEGF-A and VEGF-C sequences encoding the receptor bindingdomain. The predicted nucleotide and amino acid sequences for all 512hybrid molecules are set forth in the sequence listing as summarized inTable 2.5. TABLE 2.5 Predicted Predicted Protein VEGF-A/VEGF-C* DNASequence Sequence Chimera Seq ID NO: Seq ID NO: AAAAAAAAA 176 177CAAAAAAAA 178 179 ACAAAAAAA 180 181 CCAAAAAAA 182 183 AACAAAAAA 184 185CACAAAAAA 186 187 ACCAAAAAA 188 189 CCCAAAAAA 190 191 AAACAAAAA 192 193CAACAAAAA 194 195 ACACAAAAA 196 197 CCACAAAAA 198 199 AACCAAAAA 200 201CACCAAAAA 202 203 ACCCAAAAA 204 205 CCCCAAAAA 206 207 AAAACAAAA 208 209CAAACAAAA 210 211 ACAACAAAA 212 213 CCAACAAAA 214 215 AACACAAAA 216 217CACACAAAA 218 219 ACCACAAAA 220 221 CCCACAAAA 222 223 AAACCAAAA 224 225CAACCAAAA 226 227 ACACCAAAA 228 229 CCACCAAAA 230 231 AACCCAAAA 232 233CACCCAAAA 234 235 ACCCCAAAA 236 237 CCCCCAAAA 238 239 AAAAACAAA 240 241CAAAACAAA 242 243 ACAAACAAA 244 245 CCAAACAAA 246 247 AACAACAAA 248 249CACAACAAA 250 251 ACCAACAAA 252 253 CCCAACAAA 254 255 AAACACAAA 256 257CAACACAAA 258 259 ACACACAAA 260 261 CCACACAAA 262 263 AACCACAAA 264 265CACCACAAA 266 267 ACCCACAAA 268 269 CCCCACAAA 270 271 AAAACCAAA 272 273CAAACCAAA 274 275 ACAACCAAA 276 277 CCAACCAAA 278 279 AACACCAAA 280 281CACACCAAA 282 283 ACCACCAAA 284 285 CCCACCAAA 286 287 AAACCCAAA 288 289CAACCCAAA 290 291 ACACCCAAA 292 293 CCACCCAAA 294 295 AACCCCAAA 296 297CACCCCAAA 298 299 ACCCCCAAA 300 301 CCCCCCAAA 302 303 AAAAAACAA 304 305CAAAAACAA 306 307 ACAAAACAA 308 309 CCAAAACAA 310 311 AACAAACAA 312 313CACAAACAA 314 315 ACCAAACAA 316 317 CCCAAACAA 318 319 AAACAACAA 320 321CAACAACAA 322 323 ACACAACAA 324 325 CCACAACAA 326 327 AACCAACAA 328 329CACCAACAA 330 331 ACCCAACAA 332 333 CCCCAACAA 334 335 AAAACACAA 336 337CAAACACAA 338 339 ACAACACAA 340 341 CCAACACAA 342 343 AACACACAA 344 345CACACACAA 346 347 ACCACACAA 348 349 CCCACACAA 350 351 AAACCACAA 352 353CAACCACAA 354 355 ACACCACAA 356 357 CCACCACAA 358 359 AACCCACAA 360 361CACCCACAA 362 363 ACCCCACAA 364 365 CCCCCACAA 366 367 AAAAACCAA 368 369CAAAACCAA 370 371 ACAAACCAA 372 373 CCAAACCAA 374 375 AACAACCAA 376 377CACAACCAA 378 379 ACCAACCAA 380 381 CCCAACCAA 382 383 AAACACCAA 384 385CAACACCAA 386 387 ACACACCAA 388 389 CCACACCAA 390 391 AACCACCAA 392 393CACCACCAA 394 395 ACCCACCAA 396 397 CCCCACCAA 398 399 AAAACCCAA 400 401CAAACCCAA 402 403 ACAACCCAA 404 405 CCAACCCAA 406 407 AACACCCAA 408 409CACACCCAA 410 411 ACCACCCAA 412 413 CCCACCCAA 414 415 AAACCCCAA 416 417CAACCCCAA 418 419 ACACCCCAA 420 421 CCACCCCAA 422 423 AACCCCCAA 424 425CACCCCCAA 426 427 ACCCCCCAA 428 429 CCCCCCCAA 430 431 AAAAAAACA 432 433CAAAAAACA 434 435 ACAAAAACA 436 437 CCAAAAACA 438 439 AACAAAACA 440 441CACAAAACA 442 443 ACCAAAACA 444 445 CCCAAAACA 446 447 AAACAAACA 448 449CAACAAACA 450 451 ACACAAACA 452 453 CCACAAACA 454 455 AACCAAACA 456 457CACCAAACA 458 459 ACCCAAACA 460 461 CCCCAAACA 462 463 AAAACAACA 464 465CAAACAACA 466 467 ACAACAACA 468 469 CCAACAACA 470 471 AACACAACA 472 473CACACAACA 474 475 ACCACAACA 476 477 CCCACAACA 478 479 AAACCAACA 480 481CAACCAACA 482 483 ACACCAACA 484 485 CCACCAACA 486 487 AACCCAACA 488 489CACCCAACA 490 491 ACCCCAACA 492 493 CCCCCAACA 494 495 AAAAACACA 496 497CAAAACACA 498 499 ACAAACACA 500 501 CCAAACACA 502 503 AACAACACA 504 505CACAACACA 506 507 ACCAACACA 508 509 CCCAACACA 510 511 AAACACACA 512 513CAACACACA 514 515 ACACACACA 516 517 CCACACACA 518 519 AACCACACA 520 521CACCACACA 522 523 ACCCACACA 524 525 CCCCACACA 526 527 AAAACCACA 528 529CAAACCACA 530 531 ACAACCACA 532 533 CCAACCACA 534 535 AACACCACA 536 537CACACCACA 538 539 ACCACCACA 540 541 CCCACCACA 542 543 AAACCCACA 544 545CAACCCACA 546 547 ACACCCACA 548 549 CCACCCACA 550 551 AACCCCACA 552 553CACCCCACA 554 555 ACCCCCACA 556 557 CCCCCCACA 558 559 AAAAAACCA 560 561CAAAAACCA 562 563 ACAAAACCA 564 565 CCAAAACCA 566 567 AACAAACCA 568 569CACAAACCA 570 571 ACCAAACCA 572 573 CCCAAACCA 574 575 AAACAACCA 576 577CAACAACCA 578 579 ACACAACCA 580 581 CCACAACCA 582 583 AACCAACCA 584 585CACCAACCA 586 587 ACCCAACCA 588 589 CCCCAACCA 590 591 AAAACACCA 592 593CAAACACCA 594 595 ACAACACCA 596 597 CCAACACCA 598 599 AACACACCA 600 601CACACACCA 602 603 ACCACACCA 604 605 CCCACACCA 606 607 AAACCACCA 608 609CAACCACCA 610 611 ACACCACCA 612 613 CCACCACCA 614 615 AACCCACCA 616 617CACCCACCA 618 619 ACCCCACCA 620 621 CCCCCACCA 622 623 AAAAACCCA 624 625CAAAACCCA 626 627 ACAAACCCA 628 629 CCAAACCCA 630 631 AACAACCCA 632 633CACAACCCA 634 635 ACCAACCCA 636 637 CCCAACCCA 638 639 AAACACCCA 640 641CAACACCCA 642 643 ACACACCCA 644 645 CCACACCCA 646 647 AACCACCCA 648 649CACCACCCA 650 651 ACCCACCCA 652 653 CCCCACCCA 654 655 AAAACCCCA 656 657CAAACCCCA 658 659 ACAACCCCA 660 661 CCAACCCCA 662 663 AACACCCCA 664 665CACACCCCA 666 667 ACCACCCCA 668 669 CCCACCCCA 670 671 AAACCCCCA 672 673CAACCCCCA 674 675 ACACCCCCA 676 677 CCACCCCCA 678 679 AACCCCCCA 680 681CACCCCCCA 682 683 ACCCCCCCA 684 685 CCCCCCCCA 686 687 AAAAAAAAC 688 689CAAAAAAAC 690 691 ACAAAAAAC 692 693 CCAAAAAAC 694 695 AACAAAAAC 696 697CACAAAAAC 698 699 ACCAAAAAC 700 701 CCCAAAAAC 702 703 AAACAAAAC 704 705CAACAAAAC 706 707 ACACAAAAC 708 709 CCACAAAAC 710 711 AACCAAAAC 712 713CACCAAAAC 714 715 ACCCAAAAC 716 717 CCCCAAAAC 718 719 AAAACAAAC 720 721CAAACAAAC 722 723 ACAACAAAC 724 725 CCAACAAAC 726 727 AACACAAAC 728 729CACACAAAC 730 731 ACCACAAAC 732 733 CCCACAAAC 734 735 AAACCAAAC 736 737CAACCAAAC 738 739 ACACCAAAC 740 741 CCACCAAAC 742 743 AACCCAAAC 744 745CACCCAAAC 746 747 ACCCCAAAC 748 749 CCCCCAAAC 750 751 AAAAACAAC 752 753CAAAACAAC 754 755 ACAAACAAC 756 757 CCAAACAAC 758 759 AACAACAAC 760 761CACAACAAC 762 763 ACCAACAAC 764 765 CCCAACAAC 766 767 AAACACAAC 768 769CAACACAAC 770 771 ACACACAAC 772 773 CCACACAAC 774 775 AACCACAAC 776 777CACCACAAC 778 779 ACCCACAAC 780 781 CCCCACAAC 782 783 AAAACCAAC 784 785CAAACCAAC 786 787 ACAACCAAC 788 789 CCAACCAAC 790 791 AACACCAAC 792 793CACACCAAC 794 795 ACCACCAAC 796 797 CCCACCAAC 798 799 AAACCCAAC 800 801CAACCCAAC 802 803 ACACCCAAC 804 805 CCACCCAAC 806 807 AACCCCAAC 808 809CACCCCAAC 810 811 ACCCCCAAC 812 813 CCCCCCAAC 814 815 AAAAAACAC 816 817CAAAAACAC 818 819 ACAAAACAC 820 821 CCAAAACAC 822 823 AACAAACAC 824 825CACAAACAC 826 827 ACCAAACAC 828 829 CCCAAACAC 830 831 AAACAACAC 832 833CAACAACAC 834 835 ACACAACAC 836 837 CCACAACAC 838 839 AACCAACAC 840 841CACCAACAC 842 843 ACCCAACAC 844 845 CCCCAACAC 846 847 AAAACACAC 848 849CAAACACAC 850 851 ACAACACAC 852 853 CCAACACAC 854 855 AACACACAC 856 857CACACACAC 858 859 ACCACACAC 860 861 CCCACACAC 862 863 AAACCACAC 864 865CAACCACAC 866 867 ACACCACAC 868 869 CCACCACAC 870 871 AACCCACAC 872 873CACCCACAC 874 875 ACCCCACAC 876 877 CCCCCACAC 878 879 AAAAACCAC 880 881CAAAACCAC 882 883 ACAAACCAC 884 885 CCAAACCAC 886 887 AACAACCAC 888 889CACAACCAC 890 891 ACCAACCAC 892 893 CCCAACCAC 894 895 AAACACCAC 896 897CAACACCAC 898 899 ACACACCAC 900 901 CCACACCAC 902 903 AACCACCAC 904 905CACCACCAC 906 907 ACCCACCAC 908 909 CCCCACCAC 910 911 AAAACCCAC 912 913CAAACCCAC 914 915 ACAACCCAC 916 917 CCAACCCAC 918 919 AACACCCAC 920 921CACACCCAC 922 923 ACCACCCAC 924 925 CCCACCCAC 926 927 AAACCCCAC 928 929CAACCCCAC 930 931 ACACCCCAC 932 933 CCACCCCAC 934 935 AACCCCCAC 936 937CACCCCCAC 938 939 ACCCCCCAC 940 941 CCCCCCCAC 942 943 AAAAAAACC 944 945CAAAAAACC 946 947 ACAAAAACC 948 949 CCAAAAACC 950 951 AACAAAACC 952 953CACAAAACC 954 955 ACCAAAACC 956 957 CCCAAAACC 958 959 AAACAAACC 960 961CAACAAACC 962 963 ACACAAACC 964 965 CCACAAACC 966 967 AACCAAACC 968 969CACCAAACC 970 971 ACCCAAACC 972 973 CCCCAAACC 974 975 AAAACAACC 976 977CAAACAACC 978 979 ACAACAACC 980 981 CCAACAACC 982 983 AACACAACC 984 985CACACAACC 986 987 ACCACAACC 988 989 CCCACAACC 990 991 AAACCAACC 992 993CAACCAACC 994 995 ACACCAACC 996 997 CCACCAACC 998 999 AACCCAACC 10001001 CACCCAACC 1002 1003 ACCCCAACC 1004 1005 CCCCCAACC 1006 1007AAAAACACC 1008 1009 CAAAACACC 1010 1011 ACAAACACC 1012 1013 CCAAACACC1014 1015 AACAACACC 1016 1017 CACAACACC 1018 1019 ACCAACACC 1020 1021CCCAACACC 1022 1023 AAACACACC 1024 1025 CAACACACC 1026 1027 ACACACACC1028 1029 CCACACACC 1030 1031 AACCACACC 1032 1033 CACCACACC 1034 1035ACCCACACC 1036 1037 CCCCACACC 1038 1039 AAAACCACC 1040 1041 CAAACCACC1042 1043 ACAACCACC 1044 1045 CCAACCACC 1046 1047 AACACCACC 1048 1049CACACCACC 1050 1051 ACCACCACC 1052 1053 CCCACCACC 1054 1055 AAACCCACC1056 1057 CAACCCACC 1058 1059 ACACCCACC 1060 1061 CCACCCACC 1062 1063AACCCCACC 1064 1065 CACCCCACC 1066 1067 ACCCCCACC 1068 1069 CCCCCCACC1070 1071 AAAAAACCC 1072 1073 CAAAAACCC 1074 1075 ACAAAACCC 1076 1077CCAAAACCC 1078 1079 AACAAACCC 1080 1081 CACAAACCC 1082 1083 ACCAAACCC1084 1085 CCCAAACCC 1086 1087 AAACAACCC 1088 1089 CAACAACCC 1090 1091ACACAACCC 1092 1093 CCACAACCC 1094 1095 AACCAACCC 1096 1097 CACCAACCC1098 1099 ACCCAACCC 1100 1101 CCCCAACCC 1102 1103 AAAACACCC 1104 1105CAAACACCC 1106 1107 ACAACACCC 1108 1109 CCAACACCC 1110 1111 AACACACCC1112 1113 CACACACCC 1114 1115 ACCACACCC 1116 1117 CCCACACCC 1118 1119AAACCACCC 1120 1121 CAACCACCC 1122 1123 ACACCACCC 1124 1125 CCACCACCC1126 1127 AACCCACCC 1128 1129 CACCCACCC 1130 1131 ACCCCACCC 1132 1133CCCCCACCC 1134 1135 AAAAACCCC 1136 1137 CAAAACCCC 1138 1139 ACAAACCCC1140 1141 CCAAACCCC 1142 1143 AACAACCCC 1144 1145 CACAACCCC 1146 1147ACCAACCCC 1148 1149 CCCAACCCC 1150 1151 AAACACCCC 1152 1153 CAACACCCC1154 1155 ACACACCCC 1156 1157 CCACACCCC 1158 1159 AACCACCCC 1160 1161CACCACCCC 1162 1163 ACCCACCCC 1164 1165 CCCCACCCC 1166 1167 AAAACCCCC1168 1169 CAAACCCCC 1170 1171 ACAACCCCC 1172 1173 CCAACCCCC 1174 1175AACACCCCC 1176 1177 CACACCCCC 1178 1179 ACCACCCCC 1180 1181 CCCACCCCC1182 1183 AAACCCCCC 1184 1185 CAACCCCCC 1186 1187 ACACCCCCC 1188 1189CCACCCCCC 1190 1191 AACCCCCCC 1192 1193 CACCCCCCC 1194 1195 ACCCCCCCC1196 1197 CCCCCCCCC 1198 1199*Construct nomenclature is identical to nomenclature of Table 3.

Assembly of the hybrid molecules can also be accomplished in fewerligation steps than outlined above. For example, ligation of N123, N45,C67 and C89 can be completed in a single ligation reaction. By designingfragments with cohesive ends that are perfect complements only withcohesive ends of adjacent fragments, it is possible to ligate multiplefragments in a correct order in a single ligation reaction.

EXAMPLE 2 Expression of the Hybrid Molecules

Each of the 512 constructs were separately transfected transiently into293T cells to express the different hybrid constructs. 293T cells weregrown according to standard protocols in medium consisting of Dulbecco'smodified Eagle's medium (D-MEM), and 10% fetal bovine serum (FBS).Twenty-four hours prior to transfection, confluent dishes were diluted1:10 with fresh media into 6 wells. Four hours prior to transfection,the medium was changed. For each construct, 3 ug of DNA was transfectedusing standard protocols for calcium phosphate-mediated transfection[Sambrook et al., Molecular Cloning: A Laboratory Manual pp. 16.33-16.36(1989)]. Twenty hours post-transfection, cells were washed twice withwarm PBS and 2 ml of medium was added to each well.

Initial experiments were conducted to determine if the transfected cellswere expressing the hybrid VEGF polypeptides encoded by the hybrid DNAmolecules. Thus, 48 hours post-transfection, metabolic labeling with³⁵S-methionine and ³⁵S-cysteine was initiated using 1.3 ml/well oflabeling medium composed of MEM deficient for cysteine and methionine,0.1% BSA, 24 μCi ³⁵S-methionine-cysteine/ml (Redivue PRO-MIX, Amersham).The cell supernatant was harvested 72-78 hours post-transfection,cleared by centrifugation, and stored at 4° C.

The supernatant was immunoprecipitated with anti-pentahistidine antibody(Qiagen) by mixing 175 μl of sample supernatant with 100 μl IP mix (PBSwith 1.5% BSA, 0.05% Tween 20, and 12 μl/ml anti-pentahistidineantibody) at 4° C. overnight, with agitation. (The pSecTag I expressionvector was engineered to express each of the hybrid VEGF proteins with apolyhistidine tag.) To collect immunoprecipitated protein, 50 μl of a30% protein A sepharose (PAS, Pharmacia) slurry in PBS was added andincubated under agitation for at least 1.5 hr at 4° C. Standard bufferwas added to each immunoprecipitation sample and boiled for 5 minutes at95° C. during which the immunoprecipitated proteins become dissociatedfrom the protein A sepharose. After centrifugation, 10 μl of each samplewas analyzed on 15% SDS-PAGE under reducing conditions. The gels weredried and exposed for either 12 hours on phosphorimager plates or 4weeks on X-ray film. Results of these experiments are shown in Table 3below, in the column marked “EXP” for expression. As shown in the tablewith “Yes”, initial attempts to express the vast majority of the hybridconstructs were successful. Constructs for which weak (“weak”), and noexpression (“none”) were observed in preliminary studies also areindicated. The failure to achieve expression in initial studies isreported for completeness, and not intended to reflect a conclusion ofnon-viability or other identified problems. However, it is noteworthythat of the non-expressed constructs, almost all were those chimericmolecules in which fragment 3 was derived from VEGF-A and fragment 7 wasderived from VEGF-C. Analysis of the physical relationship between thesetwo fragments shows that residues from these two fragments barelycontact each other at the atomic level as judged from the VEGF-A crystalstructure. The incompatibility of fragment 3 from VEGF-A and fragment 7from VEGF-C may arise from incorrect folding of the molecule, perhapscaused in part by rapid glycosylation of the VEGF-C-derived fragment 7when the molecule appears in the endoplasmic reticulum. Theglycosylation sites within VEGF-A are located at a distance from thereceptor binding domain, whereas the glycosylation sites within VEGF-Care positioned closer towards the region of the molecule formed byfragment 3 that is predicted to form contacts with the third domain ofthe receptor. The carbohydrate residues may also be involved in theinteraction between ligand and receptor.

EXAMPLE 3 Binding Assays of Hybrid Molecules to Soluble VEGF Receptor-FcFusion Proteins

The hybrid proteins that were expressed in 293T cells (see Example 2 andTable 1) were tested for the ability to bind soluble VEGF receptor-Fcfusion proteins. Binding of the hybrid proteins to all three VEGFreceptors, VEGFR-1, VEGFR-2, and VEGFR-3, was analyzed in this manner.Exemplary binding assays have been described in Achen et al., Proc NatlAcad Sci USA 95:548-53 (1998), incorporated by reference in itsentirety.

It will be appreciated that binding assays can be performed with anyform of naturally occurring VEGF receptors that retain the ability tobind their respective ligands, including but not limited to whole cellsthat naturally express a receptor or that have been recombinantlymodified to express the receptor; truncated, solubilized extracellularligand binding domains of receptors; fusions comprising receptorextracellular domains fused to other proteins such as alkalinephosphatase (e.g., VEGFR-2-AP described in Cao et al., J. Biol. Chem.271:3154-62 (1996)) or immunoglobulin sequences; and fusions comprisingreceptor extracellular domains fused to tag sequences (e.g., apolyhistidine tag) useful for capturing the protein with an antibody orwith a solid support; and receptor extracellular domains chemicallyattached to solid supports such as CNBr-activated Sepharose beads.

For the present experiments, receptor binding was assayed usingconstructs comprising the extracellular domain of VEGFR-1, VEGFR-2, orVEGFR-3 fused to immunoglobulin constant region chains. The first threeIg domains of VEGFR-1 were fused to the Fc fragment from theSignal-pIgPlus vector (Ingenius/Novagen/R&D Systems). This construct(VEGFR-1-Fc) was stably expressed in Drosophila Schneider 2 (S2) cells,and purified using Protein A sepharose. Purity was analyzed by silverstaining of a PAGE gel and the functionality of the fusion protein wastested by its ability to bind ³⁵S-labeled VEGF protein. The VEGFR-2-Fcreceptor comprises the first 3 Ig domains of VEGFR-2 (encoded bynucleotides 64-972 of GenBank Acc. No. X61656) fused to the Fc fragmentin the pIg vector. The VEGFR-3-Fc receptor similarly consists if thefirst three Ig domains of VEGFR-3 (encoded by nucleotides 20-1005 ofGenBank Acc. No. X68203) fused to the Fc fragment of the pIg vector.VEGFR-2-Fc and VEGFR-3-Fc proteins were expressed in 293T cells andpurified as described above for VEGFR-1-Fc.

The binding assay procedure was identical to the immunoprecipitationusing pentahistidine antibody described in Example 2, apart from thecomposition of the immunoprecipitation (IP) mixes. The IP mixes used forthe receptor binding analysis were as follows: For VEGFR-1 bindingassays, the IP mix was phosphate buffered saline (PBS) containing 1.5%BSA, 0.06% Tween 20, 3 μg/ml heparin and 400 ng/ml VEGFR-1-Fc fusionprotein (100 μl of this IP mix was added to 200 μl of samplesupernatant); for VEGFR-2 binding assays, the IP mix was 82% conditionedcell supernatant from 293T cells transiently expressing VEGFR-2-Fcfusion protein in mixture with 18% of a PBS solution that contained 5%BSA, 0.2% Tween 20, and 10 μg/ml heparin (250 μl of IP mix was added to200 μl of sample supernatant); and for VEGFR-3 binding assays, the IPmix was 82% conditioned cell supernatant from 293T cells transientlyexpressing VEGFR-3-Fc fusion protein, 18% of PBS containing 5% BSA, 0.2%Tween 20, and 10 μg/ml heparin (250 μl of IP mix was added to 200 μl ofsample supernatant). A few selected constructs (clones 12-1, 12-5, 12-7,12-9, 12-11, 12-13, 12-14, 14-9, 23-1, 32-14, 52-15, 53-3, 82-7, 82-9,82-11, 82-13, 83-15, 84-9, and 84-11) were examined more than one time.

Results from the binding assays using ³⁵S labeled hybrid proteins aresummarized in Table 3 below. The apparent molecular weights of thedetected proteins were between 18 and 27 kD. Usually two bands werevisible with different band intensities. Sometimes, the second band wasonly detectable after long exposures. The presence of two bandscorrelates with the origin of fragment 7 and 9 of the hybrid proteinbeing examined. Fragment 7 contains a potential N-glycosylation siteirrespective of whether it was derived from VEGF-A or VEGF-C, whereasfragment 9 only contains an N-glycosylation site if it originated fromVEGF-C. Thus, the multiple bands are likely due to differentialglycosylation of the hybrid protein being analyzed. The following arepredicted bands for different combinations of glycosylation sites:

(1) fragment 7 derived from VEGF-A and fragment 9 from VEGF-A producestwo bands of ˜18 and ˜22 kD

(2) fragment 7 derived from VEGF-A and fragment 9 from VEGF-C producesan ˜26 kD band (a second band of ˜22 kD is sometimes missing, a thirdextremely weak band of ˜18 kD is sometimes visible)

(3) fragment 7 derived from VEGF-C and fragment 9 from VEGF-A producesan ˜22 kD band (a second band of ˜18 kD is sometimes missing)

(4) fragment 7 derived from VEGF-C and fragment 9 from VEGF-C producesone band of ˜23 kD.

Results of the binding assays indicate that if both glycosylation siteswere derived from VEGF-C, less heterogeneous glycosylation is observed.Molecules containing both fragment 7 from VEGF-A and fragment 9 fromVEGF-C appear to promote artificial hyperglycosylation. The VEGF-Aglycosylation site contained in fragment 7 is also prone to incompleteglycosylation

The binding assay data indicate that several of the hybrid moleculesexhibit novel binding properties. Although the analysis was notquantitative, some of the hybrid molecules show different relativesignal strengths. For example, clone 72-10 appears to have lost much ofits affinity for VEGFR-3 while retaining most of its affinity forVEGFR-2. These results suggest that among the hybrid proteins thatretained the receptor specificities of either parent protein (VEGF-A orVEGF-C), some may have undergone differential changes in their bindingaffinities towards the corresponding receptors.

In Table 3 below, column 1 lists the names of the constructs examined.Column 2 sequentially lists the 9 fragments of each construct, whereA=fragment from VEGF-A, and C=fragment from VEGF-C. Column 3 labeled“EXP” lists the results from the experiments to express the constructsin 293T cells, as described in Example 2. In this column, “none”indicates that no detectable protein was expressed; “weak” indicatesthat weak expression was detectable; and “yes” indicates that theexpressed protein was readily detectable. The final three columns listresults from the receptor binding assays described in Example 3, wherebinding to VEGFR-1-Fc; VEGFR-2-Fc; and VEGFR-3-Fc were examined. Forthese last three columns, “yes” indicates binding, “none” indicates nodetectable binding to the receptor, and “0” indicates that thisconstruct was not expressed in 293T cells, thus, could not be used forbinding assays. TABLE 3 Results of hybrid molecule expression andreceptor binding analysis VEGFR- VEGFR- VEGFR- EXP 1 2 3 31-1 A C A A AA A A A yes none none none 31-5 A C A A A A C A A none 0 0 0 31-13 A C AA A C C A A none 0 0 0 31-9 A C A A A C A A A yes none none none 31-2 AC A A A A A A C yes none none none 31-10 A C A A A C A A C yes none nonenone 31-6 A C A A A A C A C yes none none none 31-14 A C A A A C C A Cnone 0 0 0 31-12 A C A A A C A C A yes none none none 31-11 A C A A A CA C A yes none none none 31-7 A C A A A A C C A none 0 0 0 31-4 A C A AA A A C C yes none none none 31-8 A C A A A A C C C yes 0 0 0 31-3 A C AA A A A C A yes none none none 31-15 A C A A A C C C A none 0 0 0 31-16A C A A A C C C C none 0 0 0 21-1 C C C A A A A A A yes none none none21-2 C C C A A A C A A yes none none none 21-3 C C C A A C A A A yesnone none none 21-4 C C C A A C C A A yes none none none 21-5 C C C A AA A A C yes none none none 21-6 C C C A A A C A C yes none none none21-7 C C C A A C A A C yes none none none 21-8 C C C A A C C A C yesnone none none 21-9 C C C A A A A C A yes none none none 21-10 C C C A AA C C A yes none none none 21-11 C C C A A C A C A yes none none none21-12 C C C A A C C C A yes none none none 21-13 C C C A A A A C C yesnone none none 21-14 C C C A A A C C C yes none none none 21-15 C C C AA C A C C yes none none none 21-16 C C C A A C C C C yes none none none22-1 C C C C C A A A A yes none none none 22-2 C C C C C A C A A yesnone yes yes 22-3 C C C C C C A A A yes none none yes 22-4 C C C C C C CA A yes none yes yes 22-5 C C C C C A A A C yes none none none 22-6 C CC C C A C A C yes none yes yes 22-7 C C C C C C A A C yes none none none22-8 C C C C C C C A C yes none yes yes 22-9 C C C C C A A C A yes nonenone none 22-10 C C C C C A C C A yes none yes yes 22-11 C C C C C C A CA yes none none none 22-12 C C C C C C C C A yes none yes yes 22-13 C CC C C A A C C yes none none none 22-14 C C C C C A C C C yes none yesyes 22-15 C C C C C C A C C yes none none none 22-16 C C C C C C C C Cyes none yes yes 72-1 A C C C C A A A A yes none none none 72-2 A C C CC A C A A yes none yes yes 72-3 A C C C C C A A A yes none none none72-4 A C C C C C C A A yes none yes yes 72-5 A C C C C A A A C yes nonenone none 72-6 A C C C C A C A C yes none none yes 72-7 A C C C C C A AC yes none none none 72-8 A C C C C C C A C yes none yes yes 72-9 A C CC C A A C A yes none none none 72-10 A C C C C A C C A yes none yes yes72-11 A C C C C C A C A yes none none none 72-12 A C C C C C C C A yesnone yes yes 72-13 A C C C C A A C C yes none none none 72-14 A C C C CA C C C yes none yes yes 72-15 A C C C C C A C C yes none none none72-16 A C C C C C C C C yes none yes yes 11-1 A A A A A A A A A yes yesyes none 11-2 A A A A A A C A A none 0 0 0 11-3 A A A A A C A A A yesyes yes none 11-4 A A A A A C C A A none 0 0 0 11-5 A A A A A A A A Cyes yes yes none 11-6 A A A A A A C A C none 0 0 0 11-7 A A A A A C A AC yes yes yes none 11-8 A A A A A C C A C none 0 0 0 11-9 A A A A A A AC A yes yes none none 11-10 A A A A A A C C A none 0 0 0 11-11 A A A A AC A C A yes yes yes none 11-12 A A A A A C C C A none 0 0 0 11-13 A A AA A A A C C yes yes none none 11-14 A A A A A A C C C none 0 0 0 11-15 AA A A A C A C C yes yes yes none 11-16 A A A A A C C C C none 0 0 0 12-1A A A C C A A A A yes yes yes yes 12-2 A A A C C A C A A none 0 0 0 12-3A A A C C C A A A yes yes yes none 12-4 A A A C C C C A A none 0 0 012-5 A A A C C A A A C yes yes none none 12-6 A A A C C A C A C none 0 00 12-7 A A A C C C A A C yes yes yes yes 12-8 A A A C C C C A C none 0 00 12-9 A A A C C A A C A yes yes none yes 12-10 A A A C C A C C A none 00 0 12-11 A A A C C C A C A yes yes yes yes 12-12 A A A C C C C C A none0 0 0 12-13 A A A C C A A C C yes yes none yes 12-14 A A A C C A C C Cyes none none yes 12-15 A A A C C C A C C yes none yes yes 12-16 A A A CC C C C C yes none none yes 81-1 C A A A A A A A A yes yes yes none 81-2C A A A A A C A A none 0 0 0 81-3 C A A A A C A A A yes yes yes none81-4 C A A A A C C A A none 0 0 0 81-5 C A A A A A A A C yes yes yesnone 81-6 C A A A A A C A C none 0 0 0 81-7 C A A A A C A A C yes yesyes none 81-8 C A A A A C C A C none 0 0 0 81-9 C A A A A A A C A yesyes none none 81-10 C A A A A A C C A none 0 0 0 81-11 C A A A A C A C Ayes yes yes none 81-12 C A A A A C C C A none 0 0 0 81-13 C A A A A A AC C yes yes none none 81-14 C A A A A A C C C none 0 0 0 81-15 C A A A AC A C C yes yes yes none 81-16 C A A A A C C C C none 0 0 0 13-1 A A A AC A A A A yes yes yes none 13-2 A A A A C A C A A none 0 0 0 13-3 A A AA C C A A A none 0 0 0 13-4 A A A A C C C A A yes none none none 13-5 AA A A C A A A C yes yes yes none 13-6 A A A A C A C A C yes none nonenone 13-7 A A A A C C A A C yes yes yes none 13-8 A A A A C C C A C yesnone none none 13-9 A A A A C A A C A yes yes none none 13-10 A A A A CA C C A none 0 0 0 13-11 A A A A C C A C A yes yes none none 13-12 A A AA C C C C A none 0 0 0 13-13 A A A A C A A C C yes yes none none 13-14 AA A A C A C C C yes none none none 13-15 A A A A C C A C C yes yes nonenone 13-16 A A A A C C C C C none 0 0 0 14-1 A A A C A A A A A yes yesnone none 14-2 A A A C A A C A A none 0 0 0 14-3 A A A C A C A A A yesyes yes none 14-4 A A A C A C C A A none 0 0 0 14-5 A A A C A A A A Cyes yes none none 14-6 A A A C A A C A C none 0 0 0 14-7 A A A C A C A AC yes none yes none 14-8 A A A C A C C A C none 0 0 0 14-9 A A A C A A AC A yes yes yes yes 14-10 A A A C A A C C A none 0 0 0 14-11 A A A C A CA C A none 0 0 0 14-12 A A A C A C C C A none 0 0 0 14-13 A A A C A A AC C yes none none none 14-14 A A A C A A C C C none 0 0 0 14-15 A A A CA C A C C yes none none none 14-16 A A A C A C C C C none 0 0 0 23-1 C CC A C A A A A yes none none none 23-2 C C C A C A C A A yes none nonenone 23-3 C C C A C C A A A yes none none none 23-4 C C C A C C C A Ayes none none none 23-5 C C C A C A A A C yes none none none 23-6 C C CA C A C A C yes none none none 23-7 C C C A C C A A C yes none none none23-8 C C C A C C C A C yes none none none 23-9 C C C A C A A C A yesnone none none 23-10 C C C A C A C C A yes none yes none 23-11 C C C A CC A C A yes none none none 23-12 C C C A C C C C A yes none yes none23-13 C C C A C A A C C yes none none none 23-14 C C C A C A C C C yesnone yes none 23-15 C C C A C C A C C yes none none none 23-16 C C C A CC C C C yes none none none 33-1 A C A A C A A A A yes none yes none 33-2A C A A C A C A A yes none none none 33-3 A C A A C C A A A yes none yesnone 33-4 A C A A C C C A A yes none none none 33-5 A C A A C A A A Cyes none none none 33-6 A C A A C A C A C yes none none none 33-7 A C AA C C A A C yes none none none 33-8 A C A A C C C A C yes none none none33-9 A C A A C A A C A yes none yes none 33-10 A C A A C A C C A none 00 0 33-11 A C A A C C A C A yes none none none 33-12 A C A A C C C C Anone 0 0 0 33-13 A C A A C A A C C yes none none none 33-14 A C A A C AC C C yes none none none 33-15 A C A A C C A C C yes none none none33-16 A C A A C C C C C yes none none none 34-1 A C A C A A A A A yesnone none none 34-2 A C A C A A C A A none 0 0 0 34-3 A C A C A C A A Ayes none none none 34-4 A C A C A C C A A none 0 0 0 34-5 A C A C A A AA C yes none none none 34-6 A C A C A A C A C none 0 0 0 34-7 A C A C AC A A C yes none none none 34-8 A C A C A C C A C none 0 0 0 34-9 A C AC A A A C A yes none none none 34-10 A C A C A A C C A none 0 0 0 34-11A C A C A C A C A yes none none none 34-12 A C A C A C C C A none 0 0 034-13 A C A C A A A C C yes none none none 34-14 A C A C A A C C C yesnone none none 34-15 A C A C A C A C C yes none none none 34-16 A C A CA C C C C none 0 0 0 41-1 C A C A A A A A A yes yes none none 41-2 C A CA A A C A A none 0 0 0 41-3 C A C A A C A A A yes none none none 41-4 CA C A A C C A A none 0 0 0 41-5 C A C A A A A A C yes none none none41-6 C A C A A A C A C yes none none none 41-7 C A C A A C A A C yesnone none none 41-8 C A C A A C C A C none 0 0 0 41-9 C A C A A A A C Ayes none none none 41-10 C A C A A A C C A none 0 0 0 41-11 C A C A A CA C A yes none none none 41-12 C A C A A C C C A none 0 0 0 41-13 C A CA A A A C C yes none none none 41-14 C A C A A A C C C yes none nonenone 41-15 C A C A A C A C C yes none none none 41-16 C A C A A C C C Cyes none none none 42-1 C A C C C A A A A yes none none none 42-2 C A CC C A C A A none 0 0 0 42-3 C A C C C C A A A yes none none none 42-4 CA C C C C C A A one 0 0 0 42-5 C A C C C A A A C none 0 0 0 42-6 C A C CC A C A C yes none none none 42-7 C A C C C C A A C yes none none none42-8 C A C C C C C A C yes none none none 42-9 C A C C C A A C A yesnone none none 42-10 C A C C C A C C A yes none none none 42-11 C A C CC C A C A yes none none none 42-12 C A C C C C C C A yes none none none42-13 C A C C C A A C C yes none none none 42-14 C A C C C A C C C yesnone none none 42-15 C A C C C C A C C yes none none none 42-16 C A C CC C C C C yes none none none 43-1 C A C A C A A A A yes yes none none43-2 C A C A C A C A A none 0 0 0 43-3 C A C A C C A A A yes none nonenone 43-4 C A C A C C C A A none 0 0 0 43-5 C A C A C A A A C yes nonenone none 43-6 C A C A C A C A C yes none none none 43-7 C A C A C C A AC yes none none none 43-8 C A C A C C C A C yes none none none 43-9 C AC A C A A C A yes none none none 43-10 C A C A C A C C A none 0 0 043-11 C A C A C C A C A yes none none none 43-12 C A C A C C C C A none0 0 0 43-13 C A C A C A A C C yes none none none 43-14 C A C A C A C C Cyes none none none 43-15 C A C A C C A C C yes none none none 43-16 C AC A C C C C C yes none none none 44-1 C A C C A A A A A yes none nonenone 44-2 C A C C A A C A A yes none none none 44-3 C A C C A C A A Ayes none none none 44-4 C A C C A C C A A yes none none none 44-5 C A CC A A A A C yes none none none 44-6 C A C C A A C A C none 0 0 0 44-7 CA C C A C A A C yes none none none 44-8 C A C C A C C A C yes none nonenone 44-9 C A C C A A A C A yes none none none 44-10 C A C C A A C C Ayes none none none 44-11 C A C C A C A C A yes none none none 44-12 C AC C A C C C A yes none none none 44-13 C A C C A A A C C yes none nonenone 44-14 C A C C A A C C C yes none none none 44-15 C A C C A C A C Cyes none none none 44-16 C A C C A C C C C yes none none none 54-1 C C AC A A A A A yes none none none 54-2 C C A C A A C A A none 0 0 0 54-3 CC A C A C A A A yes none none none 54-4 C C A C A C C A A none 0 0 054-5 C C A C A A A A C yes none none none 54-6 C C A C A A C A C none 00 0 54-7 C C A C A C A A C yes none none none 54-8 C C A C A C C A Cnone 0 0 0 54-9 C C A C A A A C A yes none none none 54-10 C C A C A A CC A yes none none none 54-11 C C A C A C A C A yes none none none 54-12C C A C A C C C A none 0 0 0 54-13 C C A C A A A C C yes none none none54-14 C C A C A A C C C none 0 0 0 54-15 C C A C A C A C C yes none nonenone 54-16 C C A C A C C C C none 0 0 0 64-1 A A C C A A A A A yes nonenone none 64-2 A A C C A A C A A yes none none none 64-3 A A C C A C A AA yes none none none 64-4 A A C C A C C A A yes none none none 64-5 A AC C A A A A C yes none none none 64-6 A A C C A A C A C yes none nonenone 64-7 A A C C A C A A C yes none none none 64-8 A A C C A C C A Cyes none none none 64-9 A A C C A A A C A yes none none none 64-10 A A CC A A C C A yes none none none 64-11 A A C C A C A C A yes none nonenone 64-12 A A C C A C C C A yes none none none 64-13 A A C C A A A C Cyes none none none 64-14 A A C C A A C C C yes none none none 64-15 A AC C A C A C C yes none none none 64-16 A A C C A C C C C yes none nonenone 83-1 C A A A C A A A A yes yes yes none 83-2 C A A A C A C A A none0 0 0 83-3 C A A A C C A A A yes yes yes none 83-4 C A A A C C C A Anone 0 0 0 83-5 C A A A C A A A C yes yes yes none 83-6 C A A A C A C AC yes none none none 83-7 C A A A C C A A C yes yes yes none 83-8 C A AA C C C A C none 0 0 0 83-9 C A A A C A A C A yes yes none none 83-10 CA A A C A C C A none 0 0 0 83-11 C A A A C C A C A yes yes yes none83-12 C A A A C C C C A none 0 0 0 83-13 C A A A C A A C C yes yes nonenone 83-14 C A A A C A C C C none 0 0 0 83-15 C A A A C C A C C yes yesyes none 83-16 C A A A C C C C C one 0 0 0 24-1 C C C C A A A A A yesnone none none 24-2 C C C C A A C A A yes none none none 24-3 C C C C AC A A A yes none none none 24-4 C C C C A C C A A yes none none none24-5 C C C C A A A A C yes none none none 24-6 C C C C A A C A C yesnone none none 24-7 C C C C A C A A C yes none none none 24-8 C C C C AC C A C yes none none none 24-9 C C C C A A A C A yes none none none24-10 C C C C A A C C A yes none none none 24-11 C C C C A C A C A yesnone none none 24-12 C C C C A C C C A yes none none none 24-13 C C C CA A A C C yes none none none 24-14 C C C C A A C C C yes none none none24-15 C C C C A C A C C yes none none none 24-16 C C C C A C C C C yesnone none none 32-1 A C A C C A A A A yes none none none 32-2 A C A C CA C A A none 0 0 0 32-3 A C A C C C A A A yes none none none 32-4 A C AC C C C A A none 0 0 0 32-5 A C A C C A A A C yes none none none 32-6 AC A C C A C A C yes none none none 32-7 A C A C C C A A C yes none nonenone 32-8 A C A C C C C A C yes none none none 32-9 A C A C C A A C Ayes none none yes 32-10 A C A C C A C C A none 0 0 0 32-11 A C A C C C AC A yes none none yes 32-12 A C A C C C C C A none 0 0 0 32-13 A C A C CA A C C yes none none none 32-14 A C A C C A C C C yes none none yes32-15 A C A C C C A C C yes none none yes 32-16 A C A C C C C C C yesnone none yes 51-1 C C A A A A A A A yes none none none 51-2 C C A A A AC A A yes none none none 51-3 C C A A A C A A A yes none none none 51-4C C A A A C C A A yes none none none 51-5 C C A A A A A A C yes nonenone none 51-6 C C A A A A C A C yes none none none 51-7 C C A A A C A AC yes none none none 51-8 C C A A A C C A C yes none none none 51-9 C CA A A A A C A yes none none none 51-10 C C A A A A C C A none 0 0 051-11 C C A A A C A C A yes none none none 51-12 C C A A A C C C A none0 0 0 51-13 C C A A A A A C C yes none none none 51-14 C C A A A A C C Cyes none none none 51-15 C C A A A C A C C yes none none none 51-16 C CA A A C C C C none 0 0 0 52-1 C C A C C A A A A yes none none none 52-2C C A C C A C A A none 0 0 0 52-3 C C A C C C A A A yes none none none52-4 C C A C C C C A A none 0 0 0 52-5 C C A C C A A A C yes none nonenone 52-6 C C A C C A C A C yes none none none 52-7 C C A C C C A A Cyes none none none 52-8 C C A C C C C A C yes none none none 52-9 C C AC C A A C A yes none none yes 52-10 C C A C C A C C A none 0 0 0 52-11 CC A C C C A C A yes none none yes 52-12 C C A C C C C C A none 0 0 052-13 C C A C C A A C C none 0 0 0 52-14 C C A C C A C C C yes none noneyes 52-15 C C A C C C A C C yes none none yes 52-16 C C A C C C C C Cyes none yes yes 53-1 C C A A C A A A A yes none yes none 53-2 C C A A CA C A A none 0 0 0 53-3 C C A A C C A A A yes yes yes yes 53-4 C C A A CC C A A none 0 0 0 53-5 C C A A C A A A C yes none none none 53-6 C C AA C A C A C yes none none none 53-7 C C A A C C A A C yes none yes none53-8 C C A A C C C A C yes none none none 53-9 C C A A C A A C A yesnone none none 53-10 C C A A C A C C A none 0 0 0 53-11 C C A A C C A CA yes none none none 53-12 C C A A C C C C A none 0 0 0 53-13 C C A A CA A C C yes none none none 53-14 C C A A C A C C C yes none none none53-15 C C A A C C A C C yes none none none 53-16 C C A A C C C C C yesnone none none 61-1 A A C A A A A A A yes yes none none 61-2 A A C A A AC A A yes none none none 61-3 A A C A A C A A A yes yes none none 61-4 AA C A A C C A A yes none none none 61-5 A A C A A A A A C yes none nonenone 61-6 A A C A A A C A C yes none none none 61-7 A A C A A C A A Cyes none none none 61-8 A A C A A C C A C yes none none none 61-9 A A CA A A A C A yes none none none 61-10 A A C A A A C C A yes none nonenone 61-11 A A C A A C A C A yes none none none 61-12 A A C A A C C C Ayes none none none 61-13 A A C A A A A C C yes none none none 61-14 A AC A A A C C C yes none none none 61-15 A A C A A C A C C yes none nonenone 61-16 A A C A A C C C C yes none none none 62-1 A A C C C A A A Ayes yes none none 62-2 A A C C C A C A A yes 0 0 0 62-3 A A C C C C A AA yes none none none 62-4 A A C C C C C A A yes none none none 62-5 A AC C C A A A C yes none none none 62-6 A A C C C A C A C yes none nonenone 62-7 A A C C C C A A C yes none none none 62-8 A A C C C C C A Cyes none yes none 62-9 A A C C C A A C A yes none none none 62-10 A A CC C A C C A yes none yes none 62-11 A A C C C C A C A yes none yes yes62-12 A A C C C C C C A yes none none none 62-13 A A C C C A A C C yesnone yes none 62-14 A A C C C A C C C yes none none none 62-15 A A C C CC A C C yes none none none 62-16 A A C C C C C C C yes none none none63-1 A A C A C A A A A yes yes yes none 63-2 A A C A C A C A A none 0 00 63-3 A A C A C C A A A yes none yes none 63-4 A A C A C C C A A none 00 0 63-5 A A C A C A A A C yes none none none 63-6 A A C A C A C A C yesnone yes none 63-7 A A C A C C A A C yes none yes yes 63-8 A A C A C C CA C yes none none none 63-9 A A C A C A A C A yes none none none 63-10 AA C A C A C C A yes none none none 63-11 A A C A C C A C A yes none nonenone 63-12 A A C A C C C C A yes 0 0 0 63-13 A A C A C A A C C yes nonenone none 63-14 A A C A C A C C C yes none none none 63-15 A A C A C C AC C yes none none none 63-16 A A C A C C C C C yes none none none 71-1 AC C A A A A A A yes none none none 71-2 A C C A A A C A A yes none nonenone 71-3 A C C A A C A A A yes none none none 71-4 A C C A A C C A Ayes none none none 71-5 A C C A A A A A C yes none none none 71-6 A C CA A A C A C yes none none none 71-7 A C C A A C A A C yes none none none71-8 A C C A A C C A C yes none none none 71-9 A C C A A A A C A yesnone none none 71-10 A C C A A A C C A yes none none none 71-11 A C C AA C A C A yes none none none 71-12 A C C A A C C C A yes none none none71-13 A C C A A A A C C yes none none none 71-14 A C C A A A C C C yesnone none none 71-15 A C C A A C A C C yes none none none 71-16 A C C AA C C C C yes none none none 73-1 A C C A C A A A A yes none none none73-2 A C C A C A C A A yes none none none 73-3 A C C A C C A A A yesnone none none 73-4 A C C A C C C A A yes 0 0 0 73-5 A C C A C A A A Cyes none none none 73-6 A C C A C A C A C yes none none none 73-7 A C CA C C A A C yes none yes none 73-8 A C C A C C C A C yes none none none73-9 A C C A C A A C A yes none none none 73-10 A C C A C A C C A yesnone none none 73-11 A C C A C C A C A yes none none none 73-12 A C C AC C C C A yes none none none 73-13 A C C A C A A C C yes none none none73-14 A C C A C A C C C yes none none none 73-15 A C C A C C A C C yesnone yes none 73-16 A C C A C C C C C yes none none none 74-1 A C C C AA A A A yes none none none 74-2 A C C C A A C A A yes none none none74-3 A C C C A C A A A yes none none none 74-4 A C C C A C C A A yesnone none none 74-5 A C C C A A A A C yes none none none 74-6 A C C C AA C A C yes none none none 74-7 A C C C A C A A C yes none none none74-8 A C C C A C C A C yes none yes none 74-9 A C C C A A A C A yes nonenone none 74-10 A C C C A A C C A yes none yes none 74-11 A C C C A C AC A yes none none none 74-12 A C C C A C C C A yes none yes none 74-13 AC C C A A A C C yes none none none 74-14 A C C C A A C C C yes none nonenone 74-15 A C C C A C A C C yes none none none 74-16 A C C C A C C C Cyes none none none 82-1 C A A C C A A A A yes none none none 82-2 C A AC C A C A A none none none none 82-3 C A A C C C A A A yes none nonenone 82-4 C A A C C C C A A none none none none 82-5 C A A C C A A A Cyes yes none none 82-6 C A A C C A C A C none 0 0 0 82-7 C A A C C C A AC yes yes yes yes 82-8 C A A C C C C A C none 0 0 0 82-9 C A A C C A A CA yes yes yes yes 82-10 C A A C C A C C A none 0 0 0 82-11 C A A C C C AC A yes yes yes yes 82-12 C A A C C C C C A none 0 0 0 82-13 C A A C C AA C C yes yes none yes 82-14 C A A C C A C C C yes none none yes 82-15 CA A C C C A C C yes none yes yes 82-16 C A A C C C C C C yes none noneyes 84-1 C A A C A A A A A yes yes none none 84-2 C A A C A A C A A none0 0 0 84-3 C A A C A C A A A yes yes yes none 84-4 C A A C A C C A Anone 0 0 0 84-5 C A A C A A A A C yes yes none none 84-6 C A A C A A C AC none 0 0 0 84-7 C A A C A C A A C yes none none none 84-8 C A A C A CC A C none 0 0 0 84-9 C A A C A A A C A yes yes yes yes 84-10 C A A C AA C C A none 0 0 0 84-11 C A A C A C A C A yes yes yes yes 84-12 C A A CA C C C A none 0 0 0 84-13 C A A C A A A C C none 0 0 0 84-14 C A A C AA C C C none 0 0 0 84-15 C A A C A C A C C none 0 0 0 84-16 C A A C A CC C C none 0 0 0

Receptor binding properties were analyzed only for constructs that wereexpressed. If a clone was weakly expressed, its receptor bindingproperties were analyzed only if its size allowed distinction fromendogenous VEGF-A expression, or if its amino acid composition allowedremoval of endogenous VEGF-A using monoclonal anti-VEGF-A antibodies(R&D Systems) prior to assaying receptor binding. Although the epitoperecognized by this anti-VEGF-A antibody has not been characterized, ourpreliminary results indicate that the epitope is located within one ormore of fragments 2, 3, 4, 7, or 9 of VEGF-A. Thus, antibodyprecipitation of endogenous VEGF-A was performed for all constructs inwhich fragments 2, 3, 4, 7, and 9 were derived from VEGF-C. Furthermapping of the epitope of this antibody may allow similar examination ofadditional constructs. For example, if subsequent analysis indicatesthat the epitope does not reside in fragment 2, constructs in whichfragment 2 was derived from VEGF-A can also be analyzed by this method.This procedure was performed for binding to VEGFR-1 or VEGFR-2, toassess how many low affinity binding hybrid molecules were not detecteddue to interference with endogenous VEGF. Failure to detect a signal ordetection of a weak signal in the receptor binding assays does notconclusively demonstrate lack of or low receptor binding affinity. Theintrinsic set-up of the experiment does not allow detection of lowaffinity binders of VEGFR-1 and VEGFR-2 that are weakly expressed. Thus,the binding assays may have failed to detect low affinity binders ofVEGFR-1 and VEGFR-2 for some of the hybrid proteins that were weaklyexpressed.

In this assay, apparent low receptor binding affinity of alow-level-expressed hybrid molecule could be due to heterodimerizationwith endogenous VEGF. For example, if a hybrid protein has noreceptor-affinity itself, but is able to dimerize with endogenousVEGF-A, such a heterodimer may be capable of binding one of morereceptor(s) with low affinity. Purification of chimeric polypeptides ofthe invention (e.g., using immunoaffinity chromatography with anantibody that recognizes either the myc or HA tag sequences) and usingthe purified polypeptide in receptor binding assays will resolve anyambiguities caused by endogenous VEGF-A in conditioned media.Alternatively, the hybrid proteins will be expressed in insect cells,e.g., S9 cells, to avoid contamination with endogenous VEGF-A.

Lack of expression or low level expression of a particular construct maybe due to properties of the hybrid protein itself, variations in DNAquality, or may reflect mutations in the DNA acquired duringconstruction of the hybrid clone. In the present case, all constructswere sequenced after the first ligation step, and selected clones weresequenced after the second ligation step. Analysis of these sequencesindicated that no mutations occurred during the first step, and none ofthe sequences examined after the second step of construction containedmutations. Thus, any mutations present in the final clone most probablyoccurred during the final ligation step.

Thirty-six of the 512 clones were sequenced to determine the frequencywith which constructs acquired mutations during construction of theclones that resulted in changes at the amino acid level. The constructsthat were sequenced were clones 11-1 (SEQ ID NOS: 42-43), 11-16 (SEQ IDNOS: 44-45), 22-1 (SEQ ID NOS: 46-47), 22-16 (SEQ ID NOS: 48-49), 12-1to 12-16 (SEQ ID NOS: 50-81), and 31-1 to 31-16 (SEQ ID NOS: 82-113).Only 2 of the 36 clones, 12-13 and 12-16, showed a deviation from theexpected sequence. Clone 12-16 had undergone a loss of two base pairs atthe ligation junction between N45 and C67, resulting in a frameshiftmutation after the RCG triplet of fragment C5 and a stop codon only afew codons thereafter. Clone 12-13 had acquired a point mutation whichresults in the substitution of Asp by Asn at the last C-terminal aminoacid of this hybrid protein.

From the 512 hybrid constructs examined, four were chosen for furtheranalysis including sequencing to determine if any mutations occurredduring construction of the hybrid protein and repetition of bindingassays to confirm initial results. Results from binding assays of thesefour particular hybrid proteins: constructs 12-13, 12-11, 12-9, and 12-7indicate that they show novel binding patterns that are not exhibited byknown VEGF receptor ligands. 12-9 and 12-13 show binding to VEGFR-1 andVEGFR-3 but not VEGFR-2, whereas 12-7 and 12-11 exhibit binding to allthree VEGF receptors.

These results show that it is possible by combinatorial approaches toprovide VEGF-related growth factors having modified properties. Thenovel molecules constructed in some cases have been shown to havemodified biological effects compared to their wild-type ancestors, andthus may be used in applications where specificity and fine-tuning ofbiological effects are necessary. In particular, these experimentsdemonstrate that it is possible to construct a “super-VEGF”, such asclones 12-7 and 12-11, which binds all three known VEGFRs, and thereforeshould be uniquely potent in inducing vascular growth.

EXAMPLE 4 Examination of VEGF-A and VEGF-C Receptor Binding Epitopes

The VEGF-A/VEGF-C hybrid proteins can be used to examine interactionsbetween VEGF-A or VEGF-C, and their receptors. Analysis of the resultsfrom the receptor binding assays, such as those described in Table 3 andExample 3, enable careful investigation of the receptor-binding epitopesof these two VEGF growth factors. The ability of particular hybridproteins to bind one of the VEGF receptors may be correlated with thepresence of one or more particular fragments derived from one of theparent molecules. Such data can help define the amino acid residuesimportant for binding to a specific VEGF receptor. Knowledge of theprecise receptor binding epitopes of a particular VEGF protein canfacilitate the design of inhibitory molecules useful for therapeuticpurposes.

Twenty-one VEGF residues important for interfacing with VEGFR-1 areindicated in large bold text: (SEQ ID NO: 1209)GQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPK KD

Data from the chimera experiments indicates that those residues fromfragment 2, which corresponds to the N-terminal helix and residues fromfragment 7, which corresponds to strand β5 appear particularly importantfor conferring VEGFR-1 specificity. FIG. 8 is a three-dimensional modelof the interaction of a VEGF-A dimer with two VEGFR-1 molecules. The twoVEGF-A monomers are colored in green and blue, respectively. Domain 2 ofthe two VEGFR-1 receptors are depicted in gray. Red represents thelocation of residues within the VEGF-A monomers important for VEGFR-1interfacing. These residues are clustered at the two ends of the VEGFdimer and include the N-terminal helix and part of the β5 strand.

The corresponding twenty-one residues in VEGF-C that would analogouslybe involved in interfacing VEGFR-3 are indicated below in large boldtext: (SEQ ID NO: 1210)AHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSCRC MSKLD

Interestingly, analysis of the receptor binding patterns from thechimera experiments indicates that a VEGF-C-derived fragment 4(containing the β2 strand of the molecule) is absolutely required forVEGFR-3 binding specificity. Fragments 5 (which includes the β3 strand)and 8 appear to represent two other important VEGF-C fragments forVEGFR-3 binding. The amino acid sequence of VEGF-C fragments 4 and 5 isEFGVATNTFFKPPCVSVYRCG (SEQ ID NO: 1205). The TNTFxxxP (SEQ ID NO: 1204)quintet of residues is particularly noteworthy because these residuesare conserved in human, quail, and bovine VEGF-C and human VEGF-D, allof which bind VEGFR-3. The analogous residues in human VEGF-A, whichdoes not bind VEGFR-3, differ: IEYIxxxS (SEQ ID NO: 1211). FIG. 10 is athree-dimensional model of the interaction between portions of a VEGF-Cdimer and a single VEGFR-3 molecule, extrapolated from theVEGF-A/VEGFR-1 model. Blue and green represent the two VEGF-C monomersand grey represents VEGFR-3. Fragment 5 of the green VEGF-C monomer isshown in orange and fragment 4 of the same monomer is shown in white.Residues in red are those located within fragment 4 or 5 that areprobably in contact with the receptor.

FIG. 9 is a three-dimensional model that depicts the groove formed bythe fragments that appear to be important for VEGFR-3 specificity. Thisgroove is speculated to accommodate the linker region between domain 2and 3 of the VEGFR-3 receptor. The entry and the sides of this grooveare formed by the fragments that appear to be important for conferringVEGFR-3 specificity. The green and blue indicate the two VEGF-C monomersand the gray indicates the VEGFR-3 receptor molecule. The VEGF-Cresidues that are believed to participate in binding VEGFR-3 areindicated in yellow.

Although fragments 6 and 9 are involved in interaction with the VEGFreceptors, these fragments do not appear to be involved in determiningreceptor specificity.

EXAMPLE 5 Analysis of Receptor Activation or Inhibition by the HybridVEGF Proteins

The VEGF-A/VEGF-C hybrid proteins may be used for therapeuticapplications where either activation of inhibition of one or more VEGFreceptors is desired. For example, a candidate hybrid protein can beadded to stable cell lines expressing a particular VEGF receptor whoseactivation is necessary for cell survival. Survival of the cell lineindicates that the candidate hybrid protein is able to bind and activatethat particular VEGF receptor. On the other hand, death of the cell lineindicates that the candidate hybrid protein fails to activate thereceptor. Exemplary examples of such cell-survival assays have beendescribed in International Patent Publication No. WO 98/07832 and inAchen et al., Proc Natl Acad Sci USA 95:548-553 (1998), incorporatedherein by reference. This assay employs Ba/F3-NYK-EpoR cells, which areBa/F3 pre-B cells that have been transfected with a plasmid encoding achimeric receptor consisting of the extracellular domain of VEGFR-2 andthe cytoplasmic domain of the erythropoietin receptor (EpoR). Thesecells are routinely passaged in interleukin-3 (IL-3) and will die in theabsence of IL-3. However, if signaling is induced from the cytoplasmicdomain of the chimeric receptor, these cells survive and proliferate inthe absence of IL-3. Such signaling is induced by ligands which bind tothe VEGFR-2 extracellular domain of the chimeric receptor. For example,binding of VEGF-A or VEGF-D to the VEGFR-2 extracellular domain causesthe cells to survive and proliferate in the absence of IL-3. ParentalBa/F3 cells which lack the chimeric receptor are not induced by eitherVEGF-A or VEGF-D to proliferate in the absence of IL-3, indicating thatthe responses of the Ba/F3-NYK-EpoR cells to these ligands are totallydependent on the chimeric receptor.

Candidate hybrid proteins can be tested for binding to the VEGFR-2extracellular domain and subsequent activation of the chimeric receptorby assaying cell survival in the absence of IL-3. On the other hand,hybrid proteins that interfere with the binding of VEGFR-2 ligands, suchas VEGF-A or VEGF-D, to the extracellular domain, or with the activationof the cytoplasmic domain, will cause cell death in the absence of IL-3.

Cells are cultured in the presence of IL-3 until required, then washedthree times in phosphate buffered saline (PBS), resuspended in IL-3-freecell culture medium (Dulbecco's Modified Eagle's Medium (DMEM)supplemented with fetal calf serum (10%), L-glutamine (1%), geneticin (1mg/ml), streptomycin (100 μg/ml) and penicillin (60 μg/ml)), andreplated in 72-well culture plates (Nunc, Denmark) at a density ofapproximately 1000 cells/well. To assay for receptor activity, candidatehybrid proteins are added to culture wells at final concentrations of10⁻¹⁰ to 10⁻⁵ M and incubated for 1 hour at 37□C in 10% CO₂. Forassaying the ability of the candidate hybrid protein to inhibitactivation of the VEGFR-2/EpoR receptor, recombinant VEGF-A or VEGF-D isadded to the hybrid protein-containing wells at a concentration toproduce near-maximal survival of the Ba/F3-NYK-EpoR cells (typically 500ng/ml). Positive control cultures contain either VEGF-A or VEGF-Dsupernatant alone and negative control cultures contain neither hybridprotein nor growth factor. Cells are then grown in culture for 48 hours,after which time a solution of3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 500μg/ml) is added to the cultures, and incubated for another 30 minutes.MTT is converted to a blue formazan product by mitochondria, thusstaining living cells blue. Surviving blue cells in experiments whereeither activation (hybrid protein alone) or inhibition (hybridprotein+VEGF-A or VEGF-D) was assayed are counted under a microscopewith inverted optics (100× magnification) and compared to cell survivalin the positive control (VEGF-A or VEGF-D only) wells. Cell survival isnormalized such that survival in negative controls is set to 0(typically no viable cells were seen in negative controls), whilesurvival in positive controls is set to 100% (typically 300-400cells/well).

Data is analyzed by one way analysis of variance (ANOVA), with aBonferroni multiple comparisons test carried out post-hoc to test fordifferences between individual cultures of hybrid protein alone (toassay binding and activation of the receptor), or hybrid protein+VEGF-Aor VEGF-D (to assay inhibition of receptor activation), with VEGF-A orVEGF-D alone (positive control).

Repetition of the same assay using cells transfected with differentchimeric receptors (e.g., VEGFR-3/EpoR) allows screening for activationof different VEGFRs.

VEGFR-2 (KDR) and VEGFR-3 (Flt4) Autophosphorylation Assays

As an alternative indicator of activity, the ability of a hybrid proteinto stimulate autophosphorylation of a particular VEGF receptor can alsobe examined. A candidate hybrid protein is added to cells expressing aparticular VEGF receptor. The cells are then lysed andimmunoprecipitated with anti-VEGF receptor antiserum and analyzed byWestern blotting using anti-phosphotyrosine antibodies to determinehybrid protein-induced phosphorylation of the VEGF receptor.

An expression vector comprising a polynucleotide encoding a hybrid VEGFmolecule of the invention is transfected into an appropriate host cell(e.g., 293-EBNA cells using a calcium phosphate transfection method.About 48 hours after transfection, the growth medium of the transfectedcells is changed (e.g., to DMEM medium lacking fetal calf serum) and thecells are incubated (e.g, for 36 more hours) to provide a conditionedmedium. The conditioned medium is collected and centrifuged at 5000×gfor 20 minutes, and the supernatant is concentrated.

The concentrated conditioned media is used to stimulate cells expressinga VEGF receptor. For example, PAE-KDR cells (Pajusola et al., Oncogene,9:3545-55 (1994); Waltenberger et al., J. Biol. Chem., 269:26988-26995(1994)) are grown in Ham's F12 medium-10% fetal calf serum (FCS), orconfluent NIH 3T3 cells expressing VEGFR-3 are grown in DMEM medium. Thecells are starved overnight in DMEM medium or Ham's F12 supplementedwith 0.2% bovine serum albumin (BSA), and then incubated for 5 minuteswith the unconcentrated, 2-fold, 5-fold, and/or 10-fold concentratedconditioned media. Recombinant human VEGF-A or VEGF-C and conditionedmedia from mock-transfected cells are exemplary controls. In addition toconditional media, purified hybrid polypeptide can be employed in thisor other assays described herein.

After stimulation with conditioned media, the cells are washed twicewith ice-cold Tris-Buffered Saline (TBS) containing 100 mM sodiumorthovanadate and lysed in RIPA buffer containing 1 mMphenylmethylsulfonyl fluoride (PMSF), 0.1 U/ml aprotinin and 1 mM sodiumorthovanadate. The lysates are sonicated, clarified by centrifugation at16,000×g for 20 minutes and incubated for 3-6 hours on ice with 3-5 μlof antisera specific for VEGFR-3 or VEGFR-2. Immunoprecipitates arebound to protein A-Sepharose, washed three times with RIPA buffercontaining 1 mM PMSF, 1 mM sodium orthovanadate, washed twice with 10 mMTris-HCl (pH 7.4), and subjected to SDS-PAGE using a 7% gel.Polypeptides are transferred to nitrocellulose by Western blotting andanalyzed using PY20 phosphotyrosine-specific monoclonal antibodies(Transduction Laboratories) or receptor-specific antiserum and the ECLdetection method (Amersham Corp.).

The ability of a hybrid polypeptide to stimulate autophosphorylation(detected using the anti-phosphotyrosine antibodies) is scored asstimulating the receptor. The level of stimulation observed for variousconcentrations of hybrid polypeptide, relative to known concentrationsof VEGF-A or VEGF-C, provide an indication of the potency of receptorstimulation. Polypeptides that have been shown to bind the receptor, butare incapable of stimulating receptor phosphorylation, are scored asinhibitors. Inhibitory activity can be further assayed by mixing a knownreceptor agonist such as recombinant VEGF-A or VEGF-C with either mediaalone or with concentrated conditioned media, to determine if theconcentrated conditioned media inhibits VEGF-A-mediated orVEGF-C-mediated receptor phosphorylation.

In initial experiments to study tyrosine phosphorylation of VEGFR-2 andVEGFR-3 mediated by selected hybrid molecules which bind VEGFR-2 orVEGFR-3, it was observed that all hybrid proteins tested were able toinduce phosphorylation of the receptors, however to a lesser extent thanthat mediated by VEGF-A or VEGF-C. Further examination of the expressionlevels of the hybrid proteins in the baculovirus system used to producethe proteins indicate that the proteins are not all expressed incomparable amounts. Differential expression levels of the hybridproteins may explain some of the lower activities exhibited by theseproteins in assaying their ability to stimulate tyrosine phosphorylationof VEGFR-2 and VEGFR-3. In addition, the extent of phosphorylationinduced by these hybrid molecules determined using this particular assaymay not correlate with biological activity in vivo.

EXAMPLE 6 Analysis of Receptor Binding Affinities of Hybrid Proteins

Preliminary analysis of the 512 hybrid proteins indicate that a numberof them are able to bind one of more of the VEGFRs. In addition, resultsfrom these experiments suggest that some show differential bindingaffinities to one of more VEGFRs. For these experiments, the hybridprotein is expressed in an insect cell system, e.g., S9 cells, toeliminate contamination with endogenous VEGF-A found in mammalian cells.To measure the relative binding affinities of selected hybrid proteins,an ELISA-type approach is used. For example, to examine binding affinityfor VEGFR-1, serial dilutions of competing VEGFR-1-IgG fusion proteinsand a subsaturating concentration of the candidate hybrid protein taggedwith the myc epitope is added to microtitre plates coated with VEGFR-1,and incubated until equilibrium is established. The plates are thenwashed to remove unbound proteins. Hybrid molecules that remain bound tothe VEGFR-1 coated plates are detected using an anti-myc antibodyconjugated to a readily detectable label e.g., horseradish peroxidase.Binding affinities (EC50) can be calculated as the concentration ofcompeting VEGFR-IgG fusion protein that results in half-maximal binding.These values can be compared with those obtained from analysis of VEGF-Aor VEGF-C to determine changes in binding affinity of one or more of theVEGFRs. Similarly, binding to VEGFR-2 is accomplished by using aVEGFR-2-IgG fusion protein, and binding to VEGFR-3 is determined using aVEGFR-3-IgG fusion protein.

EXAMPLE 7 Endothelial Cell Migration in Collagen Gel Mediated byVEGF-A/VEGF-C Hybrid Proteins

Both VEGF-A and VEGF-C stimulate endothelial cell migration in collagengel. The hybrid proteins of the invention are examined to determine ifthey are also capable of stimulating endothelial cell migration incollagen gel, thus providing another indicia of biological activity.Exemplary examples of such cell migration assays have been described inInternational Patent Publication No. WO 98/33917, incorporated herein byreference. Briefly, bovine capillary endothelial cells (BCE) are seededon top of a collagen layer in tissue culture plates. Conditioned mediafrom cells transfected with an expression vector producing the candidatehybrid protein is placed in wells made in collagen gel approximately 4mm away from the location of the attached BCE cells. The number of BCEcells that have migrated from the original area of attachment in thecollagen gel towards the wells containing the hybrid protein is thencounted to assess the ability of the hybrid protein to induce cellmigration.

BCE cells (Folkman et al., Proc. Natl. Acad. Sci. (USA), 76:5217-5221(1979)) are cultured as described in Pertovaara et al., J. Biol. Chem.,269:6271-74 (1994). Collagen gels are prepared by mixing type I collagenstock solution (5 mg/ml in 1 mM HCl) with an equal volume of 2×MEM and 2volumes of MEM containing 10% newborn calf serum to give a finalcollagen concentration of 1.25 mg/ml. Tissue culture plates (5 cmdiameter) are coated with about 1 mm thick layer of the solution, whichis allowed to polymerize at 37° C. BCE cells are seeded atop this layer.

For the migration assays, the cells are allowed to attach inside aplastic ring (1 cm diameter) placed on top of the first collagen layer.After 30 minutes, the ring is removed and unattached cells are rinsedaway. A second layer of collagen and a layer of growth medium (5%newborn calf serum (NCS)), solidified by 0.75% low melting point agar(FMC BioProducts, Rockland, Me.), are added. A well (3 mm diameter) ispunched through all the layers on both sides of the cell spot at adistance of 4 mm, and media containing a hybrid VEGF polypeptide (ormedia alone or media containing VEGF-A or VEGF-C to serve as controls)is pipetted daily into the wells. Photomicrographs of the cellsmigrating out from the spot edge are taken, e.g., after six days,through an Olympus CK 2 inverted microscope equipped with phase-contrastoptics. The migrating cells are counted after nuclear staining with thefluorescent dye bisbenzimide (1 mg/ml, Hoechst 33258, Sigma).

The number of cells migrating at different distances from the originalarea of attachment towards wells containing media conditioned by thenon-transfected (control) or transfected (mock; hybrid; VEGF-C; orVEGF-A) cells are determined 6 days after addition of the media. Thenumber of cells migrating out from the original ring of attachment arecounted in five adjacent 0.5 mm×0.5 mm squares using a microscope ocularlens grid and 10× magnification with a fluorescence microscope. Cellsmigrating further than 0.5 mm are counted in a similar way by moving thegrid in 0.5 mm steps.

The ability of a hybrid polypeptide to induce migration of BCE cells inindicative of receptor agonist activity. The number of migrating cellsin the presence of a hybrid protein versus a similar concentration ofVEGF-A or VEGF-C provides an indication of the potency of agonistactivity. Polypeptides that have been shown to bind the receptorsexpressed on BCE cells, but are incapable of stimulating migration, arescored as potential inhibitors. Inhibitory activity can be furtherassayed by mixing a known receptor agonist such as recombinant VEGF-A orVEGF-C with either media alone or with concentrated conditioned media,to determine if the concentrated conditioned media inhibitsVEGF-A-mediated or VEGF-C-mediated BCE migration.

EXAMPLE 8 Analysis of the Ability of Hybrid Proteins to Induce VascularPermeability

Both VEGF-A and VEGF-C are capable of increasing the permeability ofblood vessels. The hybrid proteins of the invention are assayed todetermine which of these proteins possess this biological activity andwhich inhibit it. For example, vascular permeability assays according toMiles and Miles, J. Physiol 118:228-257 (1952), incorporated herein inits entirety, are used to analyze the hybrid proteins. Briefly,following intravenous injection of a vital dye, such as pontamine skyblue, animals such as guinea pigs are injected intradermally with acomposition containing the candidate hybrid protein being examined. Forcontrols, media alone or media containing VEGF-A or VEGF-C is injectedin the same manner. After a period of time, the accumulation of dye atthe injection site on the skin is measured. Those hybrid proteins thatincrease permeability will result in greater accumulation of dye at theinjection site as compared to those hybrid proteins that fail to inducevascular permeability.

In a variation of this assay, hybrid polypeptides that are suspected ofbeing inhibitors of VEGF-A or VEGF-C are first mixed with VEGF-A or withVEGF-C at varying ratios (e.g., 50:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5,1:10) and the mixtures are injected intradermally into the animals. Inthis manner, the ability of the hybrid polypeptide to inhibitVEGF-A-mediated or VEGF-C-mediated vascular permeability is assayed.

EXAMPLE 9 Endothelial Cell Proliferation Assay

The mitogenic activity of hybrid proteins can be examined usingendothelial cell proliferation assays such as that described in Breieret al., Dev 114:521-532 (1992), incorporated herein in its entirety. Thehybrid proteins are expressed in a mammalian cell line e.g., COS cells.Culture supernatants are then collected and assayed for mitogenicactivity on bovine aortic endothelial (BAE) cells by adding thesupernatants to the BAE cells. After three days, the cells aredissociated with trypsin and counted using a cytometer to determine anyeffects of the hybrid protein on the proliferative activity of the BAEcells. As negative controls, DMEM supplemented with 10% FCS and theconditioned media from untransfected COS cells or from COS cellstransfected with vector alone can be used. Supernatants from cellstransfected with constructs expressing proteins that have been shown toinduce proliferation of BAE cells (e.g., VEGF-A) can be used as apositive control.

EXAMPLE 10 Examination of the Ability of Hybrid Proteins ExpressedThrough the Human K14 Keratin Promoter to Induce Growth of LymphaticVessels in Skin of Transgenic Mice

Experiments are conducted in transgenic mice to analyze the specificeffects of overexpression of hybrid proteins in tissues. Thephysiological effects in vivo provide an indication of receptoractivation/inhibition profile and an indication of the potentialtherapeutic action of a hybrid protein. In one variation, the human K14keratin promoter which is active in the basal cells of stratifiedsquamous epithelia [Vassar et al., Proc. Natl. Acad. Sci. (USA),86:1563-1567 (1989)], is used as the expression control element in therecombinant hybrid protein transgene. The vector containing the K14keratin promoter is described in Vassar et al., Genes Dev., 5:714-727(1991) and Nelson et al., J. Cell Biol. 97:244-251 (1983).

A DNA fragment containing the K14 promoter, hybrid protein cDNA, and K14polyadenylation signal is synthesized, isolated, and injected intofertilized oocytes of the FVB-NIH mouse strain. The injected zygotes aretransplanted to oviducts of pseudopregnant C57BL/6×DBA/2J hybrid mice.The resulting founder mice are then analyzed for the presence of thetransgene by polymerase chain reaction of tail DNA using appropriateprimers or by Southern analysis.

These transgenic mice are then examined for evidence of angiogenesis orlymphangiogenesis in the skin, such as the lymphangiogenesis seen intransgenic mice that overexpress VEGF-C [see International PublicationWO98/33917]. Histological examination of K14-VEGF-C transgenic miceshowed that in comparison to the skin of wildtype littermates, thedorsal dermis was atrophic and connective tissue was replaced by largelacunae devoid of red cells, but lined with a thin endothelial layer.These distended vessel-like structures resembled those seen in humanlymphangiomas. The number of skin adnexal organs and hair follicles werereduced. In the snout region, an increased number of vessels was alsoseen.

Examination of the vessels in the skin of the transgenic mice usingantibodies that recognize proteins specific for either blood orlymphatic vessels can further verify the identity of these vessels.Collagen types IV, XVIII [Muragaki et al., Proc. Natl. Acad. Sci. USA,92: 8763-8776 (1995)] and laminin are expressed in vascular endothelialcells while desmoplakins I and II (Progen) are expressed in lymphaticendothelial cells. See Schmelz et al., Differentiation, 57: 97-117(1994).

EXAMPLE 11 Analysis of Hybrid Proteins in Promoting or InhibitingMyelopoiesis

Overexpression of VEGF-C in the skin of K14-VEGF-C transgenic micecorrelates with a distinct alteration in leukocyte populations [seeInternational Publication WO98/33917]. Notably, the measured populationsof neutrophils were markedly increased in the transgenic mice. Theeffects of the hybrid proteins on hematopoiesis can be analyzed usingfluorescence-activated cell sorting analysis using antibodies thatrecognize proteins expressed on specific leukocyte cell populations.Leukocytes populations are analyzed in blood samples taken from the F1transgenic mice described in Example 13, and from their non-transgeniclittermates.

EXAMPLE 12 Effects of Hybrid Proteins on Growth and Differentiation ofHuman CD34+ Progenitor Cells In Vitro

Addition of VEGF-C to cultures of cord blood CD34+ cells induces cellproliferation. Co-culture of GM-CSF, IL-3, GM-CSF+IL-3, or GM-CSF+SCFwith VEGF-C leads to an enhancement of proportions of myeloid cells [seeInternational Publication WO98/33917]. Hybrid proteins of the inventioncan also be examined for their ability to induce growth of CD34+progenitor cells in vitro. Human CD34+ progenitor cells (HPC, 10×103)are isolated from bone marrow or cord blood mononuclear cells using theMACS CD34 Progenitor cell Isolation Kit (Miltenyi Biotec, BergishGladbach, Germany), according to the instructions of the manufacturerand cultured in RPMI 1640 medium supplemented with L-glutamine (2.5 mM),penicillin (125 IE/ml), streptomycin (125 μg/ml) and pooled 10%umbilical cord blood (CB) plasma at 37 oC in a humidified atmosphere inthe presence of 5% CO2 for seven days, with or without hybrid protein atconcentrations ranging from 10 ng/ml to 1 μg/ml. After seven days, totalcell number is evaluated in each culture.

The co-stimulatory effect of hybrid proteins in cultures eithersupplemented with recombinant human stem cell factor (rhSCF, 20 ng/mlPreproTech, Rocky Hill, N.Y.) alone or a combination of granulocytemacrophage colony stimulating factor (rhGM-CSF, 100 ng/ml, Sandoz,Basel, Switzerland) plus SCF can also be examined. Experiments can alsobe conducted to analyze the co-stimulatory effects of hybrid protein ontotal cell yields of serum-free cultures of CB CD34+ HPC cellssupplemented with either GM-CSF alone, IL-3 (rhIL-3, 100 U/ml, Behring AG, Marburg, Germany) alone; or a combination of GM-CSF plus IL-3.

Cells from the (7 day) plasma-supplemented cultures described above arealso analyzed for the expression of the early granulomonocytic markermolecules lysozyme (LZ) and myeloperoxidase (MPO) as well as thelipopolysaccharide (LPS) receptor CD14 using immunofluorescence.

In another series of experiments, CD34+ cells are cultured in mediumsupplemented with 50 ng/ml M-CSF, with or without 100 ng/ml hybridprotein, for seven days. After seven days, the cultures were analyzed todetermine the percentages of CD14+ cells and mean fluorescenceintensity.

EXAMPLE 13 Analysis of Hybrid Proteins Using CAM Assays

The chorioallantoic membrane (CAM) assay described in e.g., Oh et al.,Dev Biol 188:96-109 (1997), incorporated herein in its entirety, is acommonly used method to examine the in vivo effects of angiogenicfactors. Using this assay, VEGF growth factors including both VEGF-A andVEGF-C have been shown to induce the development of blood vessels [Oh etal., Dev Biol 188:96-109 (1997)]. Thus, this method can be used to studythe angiogenic properties of the hybrid proteins.

Briefly, on day 4 of development, a window is cut out into the eggshellof chick or quail eggs. The embryos are checked for normal development,the window in the eggshell is sealed with cellotape, and the eggs areincubated until day 13 of development. Approximately 3.3 μg of hybridprotein dissolved in 5 μl of distilled water is added to Thermanoxcoverslips (Nunc, Naperville, Ill.), which have been cut into disks withdiameters of approximately 5 mm, and air dried. Disks without addedprotein are used as controls. The dried disks are then applied on thechorioallantoic membrane (CAM) of the eggs. After 3 days, the disks areremoved and fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in0.12 M sodium cacodylate buffer. The fixed specimens are photographedand embedded in Epon resin (Serva, Germany) for semi- (0.75 μm) andultrathin (70 nm) sectioning. Both semi- and ultrathin sections are cutusing an Ultracut S (Leika, Germany). Ultrathins sections are analyzedby an EM 10 (Zeiss, Germany). Specimens are then analyzed for evidenceof growth of new capillaries, which would indicate that the hybridprotein being examined is capable of stimulating angiogenesis.

EXAMPLE 14 Analysis of Homo- or Hetero Dimerization of the VEGF-A/VEGF-CHybrid Proteins

Activation of tyrosine receptors is commonly mediated by ligand-inducedreceptor dimerization. Investigation of interactions between VEGF andVEGFR-2 indicate that receptor dimerization is accomplished via liganddimerization in which both receptors bind parts of each of the twoligand proteins that constitute the homo- or heterodimer. Mutant VEGFproteins that can bind to VEGFR-2 but are unable to dimerize, cannotactivate the receptor [Fuh et al., J Biol Chem 273:11197-11204 (1998)].All of the VEGF family members are capable of homo- and/orheterodimerization. VEGF-A and VEGF-C fail to heterodimerize with eachother. However, some of the VEGF-A/VEGF-C hybrid proteins may dimerizewith each other or with one or both of the parent molecules. The hybridproteins may also be capable of homodimerization. The followingprotocols are designed to identify dimerization capabilities of thehybrid proteins of the invention. A candidate hybrid protein isco-expressed with a different hybrid protein or one of the parentmolecules in a cell line e.g., 293T or S9 cells. Extracts from thesecells are prepared and used for immunoprecipitation using an antibodythat recognizes only one of the two proteins being examined. Theimmunoprecipitated proteins are then subjected to SDS-PAGE and analyzed.If both proteins are detected on the gel, heterodimerization occurredbetween the two proteins being examined. On the other hand, if only theprotein recognized by the antibody used during immunoprecipitation isdetected, dimerization failed to occur between the two proteins. Sincedimerization appears to be critical for receptor activation, hybridproteins that bind receptor but Feb. 16, 2000 fail to dimerize with selfor with natural VEGF growth factors endogenously expressed by cells areexpected to be inhibitors of endogenous vascular endothelial growthfactor activity.

Heterodimers comprising a polypeptide of the invention with otherpolypeptides of the invention or with naturally occurring members of theVEGF family of growth factors may be generated essentially as describedin Cao et al., J. Biol. Chem., 271:3154-62 (1996). Briefly, arecombinantly produced hybrid polypeptide is mixed at an equimolar ratiowith another recombinantly produced polypeptide of interest, such as aVEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGFα, PDGFβ, or c-fos inducedgrowth factor polypeptide. (See, e.g., Collins et al., Nature,316:748-750 (1985) (PDGF-β, GenBank Acc. No. X02811); Claesson-Welsh etal., Proc. Natl. Acad. Sci. USA, 86(13):4917-4921 (1989) (PDGF-α,GenBank Acc. No. M22734); Claesson-Welsh et al., Mol. Cell. Biol.8:3476-3486 (1988) (PDGF-β, GenBank Acc. No. M21616); Olofsson et al.,Proc. Natl. Acad. Sci. (USA), 93:2576-2581 (1996) (VEGF-B, GenBank Acc.No. U48801); Maglione et al., Proc. Natl. Acad. Sci. (USA),88(20):9267-9271 (1996) (PlGF, GenBank Acc. No. X54936); Heldin et al.,Growth Factors, 8:245-252 (1993); Folkman, Nature Med., 1:27-31 (1995);Friesel et al., FASEB J., 9:919-25 (1995); Mustonen et al., J. Cell.Biol., 129:895-98 (1995); Orlandini, S., Proc. Natl. Acad. Sci. USA,93(21):11675-11680 (1996); and others cited elsewhere herein. The mixedpolypeptides are incubated in the presence of guanidine-HCl and DTT. Thethiol groups are then protected with S-sulfonation, and the protein isdialyzed overnight, initially against urea/glutathione-SH,glutathione-S-S-glutathione, and subsequently against 20 mM Tris-HCl.

The heterodimers are screened to determine their binding affinity withrespect to receptors of the VEGF/PDGF family (especially VEGFR-1,VEGFR-2, and VEGFR-3), and their ability to stimulate the receptors(e.g., assaying for dimer-stimulated receptor phosphorylation in cellsexpressing the receptor of interest on their surface). The bindingassays may be competitive binding assays such as those described hereinand in the art. In the initial binding assays, recombinantly producedproteins comprising the extracellular domains of receptors areemployable, as described in preceding examples for VEGFR-2 and VEGFR-3.Heterodimers that bind and stimulate receptors are useful as recombinantgrowth factor polypeptides. Heterodimers that bind but do not stimulatereceptors are useful as growth factor antagonists. Heterodimers thatdisplay agonistic or antagonistic activities in the screening assays arefurther screened using, e.g., endothelial cell migration assays,vascular permeability assays, and in vivo assays. It will also beapparent from the preceding examples that dimers comprising two VEGF-Cpolypeptides (i.e., dimers of identical VEGF-C polypeptides as well asdimers of different VEGF-C polypeptides) are advantageously screened foragonistic and antagonistic activities using the same assays.

EXAMPLE 15 Determination of Biological Half-Life of the VEGF-A/VEGF-CHybrid Proteins

Knowledge of the in vivo biological half-life of a compound is valuablefor therapeutic applications. Although the biological half-life of thehybrid proteins has not been determined in vivo, preliminary results invitro indicate that the VEGF-A/VEGF-C hybrid proteins described aboveexhibit different half-lives. Incubation of cell supernatants containingspecific hybrid proteins at 4□C for approximately two months revealdifferent protein stabilities for the various hybrid proteins.Examination of the in vivo biological half-life can be determined byinjecting iodine-labeled hybrid protein into animals. Briefly, 50 μg ofhybrid protein are iodinated using IODO-GEN (Pierce) according to themanufacturer's instructions to a specific radioactivity of approximately2-10 μCi/μg protein. The iodinated protein is purified using PD-10Sephadex (Pharmacia) according to the manufacturer's instructions. 12-16week old mice (weighing 20-25 g) are anesthetized with sodiumpentobarbital (1 mg/20 g body weight mouse) during the course of theexperiment. 5-10 pmol of the radiolabeled protein diluted in 100 μlsterile saline are is injected into the tail vein over 30 seconds. Atspecific time points (1 min, 2 min, 4 min, 8 min, 15 min, 30 min, 60min, and 120 min), 40-50 μl of blood is collected by periorbitalbleeding or form the tail artery. 25 μl of the plasma fraction of eachblood sample is then spotted onto Whatman filter paper, precipitatedwith 10% trichloroacetic acid (TCA), and rinsed with ethanol. The amountof radiolabeled protein present in the plasma fraction is determined byquantifying the radioactivity using a gamma counter. Polypeptides thatdisplay improved half-life relative to that of naturally occurring VEGFsare a preferred genus of polypeptides of the invention. Polypeptidesthat show 25%, 50%, 75% or 100% improvement of half-life to that ofnaturally occurring VEGFs are highly preferred.

EXAMPLE 16 Construction of Hybrid Molecules Using Other VEGF or PDGFFamily Proteins

The procedure described in Example 1 can be extended to create hybridmolecules using any of the PDGF/VEGF growth factors. Members of thePDGF/VEGF family, which comprises at least VEGF-A (SEQ ID NOS: 1 and 2),PlGF (SEQ ID NOS: 114 and 115), VEGF-B (SEQ ID NOS: 116 and 117), VEGF-C(SEQ ID NOS: 21 and 22), VEGF-D (SEQ ID NOS: 118 and 119), VEGF-E (SEQID NOS: 120 and 121), and NZ2 VEGF (SEQ ID NOS: 122 and 123), D1701 VEGF(SEQ ID NOS: 150 and 151); NZ10 VEGF [described in SEQ ID NO: 11 ofInternational Patent Application PCT/US99/25869, incorporated herein inits entirety]; PDGF-A (SEQ ID NO: 124 and 125), PDGF-B (SEQ ID NO: 126and 127), and fallotein (SEQ ID NO: 148 & 149) share sufficient homologywith each other within the receptor binding domain to permit designingoligonucleotides with unique cohesive ends as taught in Example 1 withrespect to VEGF-A and VEGF-C. As shown by the successful results inExamples 1-3, oligonucleotides designed to provide double-strandedfragments having cohesive ends as short as 3-6 bases in length aresufficient to permit successful recombination into novel hybridmolecules (with very few unintended mutations).

While the presence of cohesive ends greatly facilitated ligation offragments in a desired order and orientation, it will be appreciatedthat ligation of fragments can also be accomplished without cohesiveends. Blunt-end fragments also can be synthesized and annealed togenerate hybrid proteins using the method described above. With ablunt-end strategy, the nucleotide sequences of the parent molecules donot need to be examined for the presence of nucleotide identity toenable the creation of cohesive ends. However, additional post-ligationscreening may be required to identify hybrids that contain fragments inthe desired order and orientation.

Using such guidelines, oligonucleotide pairs are designed and annealedas described in Example 1 to provide DNA fragments of the receptor forbinding domain of two or more VEGF proteins. Combinatorial ligation ofthe various DNA fragments produces novel hybrid polypeptides that arescreened for receptor binding and for biological properties such asability to stimulate or inhibit endothelial cell growth and migrationand modulate vascular permeability.

EXAMPLE 17 Generation of Hybrid Molecules Using PCR-Driven DNA Shuffling

The following protocol provides an alternative “DNA shuffling”methodology for generating hybrid vascular endothelial growthfactor-encoding polynucleotides and polypeptides. DNA shufflingprocedures have been described in the literature for enzymes such asantibiotic-resistance-conferring proteins, and a few other proteinfamilies. [See, e.g., Chang et al., Nature Biotechnology, 17: 793-797(1999); Kikuchi et al., Gene, 236: 159-167 (1999); Harayama et al.,TIBTECH, 16: 76-82 (1998); Crameri et al., Nature, 391: 288-291 (1998);Patten et al., Curr. Opin. Biotechnology, 8: 724-733 (1997); Zhang etal., Proc. Natl. Acad. Sci. USA, 94: 4504-09 (1997); Stemmer, Proc.Natl. Accd. Sci. USA, 91: 10747-1074 (1994); and Stemmer, Nature, 370:389-391 (1994), all incorporated herein by reference in their entirety.]

Two or more cDNAs encoding vascular endothelial growth factorpolypeptides are first cloned and amplified. In a preferred embodiment,only those portions of the cDNAs that encode minimum VEGFreceptor-binding domains, and optionally small 5′ and 3′ additionalsequences from the cDNAs, are amplified.

The purified and isolated cDNAs are digested into fragments of about10-75 base pairs using restriction endonucleases and/or DNaseI, and thefragments of this desired size range are purified and isolated (e.g., byagarose gel electrophoresis, electroelution, and ethanol precipitation).

The purified and isolated fragments from the two or more VEGFs are mixedand subjected to a self-priming polymerase chain reaction to shuffle thefragments in order to form new hybrid molecules. Exemplary PCR protocolsare set forth in Kikuchi et al. (1999) and Stemmer (1994). The annealingtemperature in the PCR reactions is adjusted based on the level ofsequence identity between the original cDNAs, to assure that annealingof heterologous sequences containing imperfect matches is possible.After conducting 25-50 cycles of PCR without primers, an aliquot fromthe PCR reaction is selected and used as template for a second round ofPCR with primers based on 5′ and 3′ sequences of the original cDNAs.Preferably, the primers also include restriction endonucleaserecognition sequences to facilitate cloning the resultant second-roundPCR products into an expression vector.

The resultant clones are ligated into an expression vector andtransformed or transected into host cells to express the novel hybridVEGF polypeptides (if any) encoded thereby. The proteins are screenedusing receptor binding and/or activity assays as set forth in thepreceding examples, to select those clones which encode polypeptideshaving desirable receptor agonist/antagonist profiles.

INDEX FOR SEQUENCE LISTING

SEQ ID NOS: 1 & 2 are the nucleotide and amino acid sequences of VEGF-A

SEQ ID NOS: 3-11 are VEGF-A forward primers

SEQ ID NOS: 12-20 are VEGF-A reverse primers

SEQ ID NOS: 21 & 22 are the nucleotide and amino acid sequences ofVEGF-C

SEQ ID NOS: 23-31 are VEGF-C forward primers

SEQ ID NOS: 32-40 are VEGF-C reverse primers

SEQ ID NO: 41 is the nucleotide sequence of pSecTagI

SEQ ID NOS: 42 & 43 are the nucleotide and amino acid sequences of clone11-1. The VEGF receptor binding domain (derived from VEGF-A and VEGF-C)correspond to amino acids 1-102 of SEQ ID NO: 43.

SEQ ID NOS: 44 & 45 are the nucleotide and amino acid sequences of clone11-16. (VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO:45).

SEQ ID NOS: 46 & 47 are the nucleotide and amino acid sequences of clone22-1. (VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 47)

SEQ ID NOS: 48 & 49 are the nucleotide and amino acid sequences of clone22-16. (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 49)

SEQ ID NOS: 50-51 are the nucleotide and amino acid sequences of clone12-1.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 51)

SEQ ID NOS: 52-53 are the nucleotide and amino acid sequences of clone12-2.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 53)

SEQ ID NOS: 54-55 are the nucleotide and amino acid sequences of clone12-3.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 55)

SEQ ID NOS: 56-57 are the nucleotide and amino acid sequences of clone12-4.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 57)

SEQ ID NOS: 58-59 are the nucleotide and amino acid sequences of clone12-5.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 59)

SEQ ID NOS: 60-61 are the nucleotide and amino acid sequences of clone12-6.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 61)

SEQ ID NOS: 62-63 are the nucleotide and amino acid sequences of clone12-7.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 63)

SEQ ID NOS: 64-65 are the nucleotide and amino acid sequences of clone12-8.

(VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO: 65)

SEQ ID NOS: 66-67 are the nucleotide and amino acid sequences of clone12-9.

(VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO: 67)

SEQ ID NOS: 68-69 are the nucleotide and amino acid sequences of clone12-10.

(VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO: 69)

SEQ ID NOS: 70-71 are the nucleotide and amino acid sequences of clone12-11.

(VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO: 71)

SEQ ID NOS: 72-73 are the nucleotide and amino acid sequences of clone12-12.

(VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO: 73)

SEQ ID NOS: 74-75 are the nucleotide and amino acid sequences of clone12-13.

(VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO: 75)

SEQ ID NOS: 76-77 are the nucleotide and amino acid sequences of clone12-14.

(VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO: 77)

SEQ ID NOS: 78-79 are the nucleotide and amino acid sequences of clone12-15.

(VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO: 79)

SEQ ID NOS: 80-81 are the nucleotide and amino acid sequences of clone12-16.

(VEGF receptor binding domain=amino acids 1-54 of SEQ ID NO: 81)

SEQ ID NOS: 82-83 are the nucleotide and amino acid sequences of clone31-1

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 83)

SEQ ID NOS: 84-85 are the nucleotide and amino acid sequences of clone31-2

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 85)

SEQ ID NOS: 86-87 are the nucleotide and amino acid sequences of clone31-3

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 87)

SEQ ID NOS: 88-89 are the nucleotide and amino acid sequences of clone31-4

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 89)

SEQ ID NOS: 90-91 are the nucleotide and amino acid sequences of clone31-5

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 91)

SEQ ID NOS: 92-93 are the nucleotide and amino acid sequences of clone31-6

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 93)

SEQ ID NOS: 94-95 are the nucleotide and amino acid sequences of clone31-7

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 95)

SEQ ID NOS: 96-97 are the nucleotide and amino acid sequences of clone31-8

(VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO: 97)

SEQ ID NOS: 98-99 are the nucleotide and amino acid sequences of clone31-9

(VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 99)

SEQ ID NOS: 100-101 are the nucleotide and amino acid sequences of clone31-10 (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 101)

SEQ ID NOS: 102-103 are the nucleotide and amino acid sequences of clone31-11 (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 103)

SEQ ID NOS: 104-105 are the nucleotide and amino acid sequences of clone31-12 (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 105)

SEQ ID NOS: 106-107 are the nucleotide and amino acid sequences of clone31-13 (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 107)

SEQ ID NOS: 108-109 are the nucleotide and amino acid sequences of clone31-14 (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 109)

SEQ ID NOS: 110-111 are the nucleotide and amino acid sequences of clone31-15 (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 111)

SEQ ID NOS: 112-113 are the nucleotide and amino acid sequences of clone31-16 (VEGF receptor binding domain=amino acids 1-105 of SEQ ID NO: 113)

SEQ ID NOS: 114 & 115 are the nucleotide and amino acid sequences ofPlGF

SEQ ID NOS: 116 & 117 are the nucleotide and amino acid sequences ofVEGF-B

SEQ ID NOS: 118 & 119 are the nucleotide and amino acid sequences ofVEGF-D

SEQ ID NOS: 120 & 121 are the nucleotide and amino acid sequences ofVEGF-E

SEQ ID NOS: 122 & 123 are the nucleotide and amino acid sequences of NZ2VEGF

SEQ ID NOS: 124 & 125 are the nucleotide and amino acid sequences ofPDGF-A

SEQ ID NOS: 126 & 127 are the nucleotide and amino acid sequences ofPDGF-B

SEQ ID NOS: 128-136 are the amino acid sequences of fragments A1-A9

SEQ ID NOS: 137-145 are the amino acid sequences of fragments C1-C9

SEQ ID NOS: 146 & 147 are the nucleotide and amino acid sequences of the232 amino acid isoform of VEGF-A

SEQ ID NOS: 148 & 149 are the nucleotide and amino acid sequences offallotein

SEQ ID NOS: 150 & 151 are the nucleotide and amino acid sequences D1701VEGF

SEQ ID NOS: 152 & 153 are the nucleotide and amino acid sequences ofclone 14-9 (VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO:153)

SEQ ID NOS: 154 & 155 are the nucleotide and amino acid sequences ofclone 23-10 (VEGF receptor binding domain=amino acids 1-105 of SEQ IDNO: 155)

SEQ ID NOS: 156 & 157 are the nucleotide and amino acid sequences ofclone 32-14 (VEGF receptor binding domain=amino acids 1-105 of SEQ IDNO: 157)

SEQ ID NOS: 158 & 159 are the nucleotide and amino acid sequences ofclone 52-15 (VEGF receptor binding domain=amino acids 1-105 of SEQ IDNO: 159)

SEQ ID NOS: 160 & 161 are the nucleotide and amino acid sequences ofclone 53-3 (VEGF receptor binding domain=amino acids 1-103 of SEQ ID NO:161)

SEQ ID NOS: 162 & 163 are the nucleotide and amino acid sequences ofclone 82-7 (VEGF receptor binding domain=amino acids 1-102 of SEQ ID NO:163)

SEQ ID NOS: 164 & 165 are the nucleotide and amino acid sequences ofclone 82-9 (VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO:165)

SEQ ID NOS: 166 & 167 are the nucleotide and amino acid sequences ofclone 82-11 (VEGF receptor binding domain=amino acids 1-104 of SEQ IDNO: 167)

SEQ ID NOS: 168 & 169 are the nucleotide and amino acid sequences ofclone 82-13 (VEGF receptor binding domain=amino acids 1-104 of SEQ IDNO: 169)

SEQ ID NOS: 170 & 171 are the nucleotide and amino acid sequences ofclone 83-15 (VEGF receptor binding domain=amino acids 1-104 of SEQ IDNO: 171)

SEQ ID NOS: 172 & 173 are the nucleotide and amino acid sequences ofclone 84-9 (VEGF receptor binding domain=amino acids 1-104 of SEQ ID NO:173)

SEQ ID NOS: 174 & 175 are the nucleotide and amino acid sequences ofclone 84-11 (VEGF receptor binding domain=amino acids 1-104 of SEQ IDNO: 175)

SEQ ID NOS: 176-1199 are indexed above in Example 1 in Table 2.5.

SEQ ID NO: 1200 is the sequence formulaP-[PS]-C-V-X(3)-R-C-[GSTA]-G-C-C.

SEQ ID NO: 1201 is the sequence formulaC-X(22-24)-P-[PSR]-C-V-X(3)-R-C-[GSTA]-G-C-C-X(6)-C-X(32-41)-C.

SEQ ID NO: 1202 is the sequence formulaC-X(18-28)-P-X-C-X(4)-R-C-X-G-C(1-2)-X(6-12)-C-X(30-46)-C.

SEQ ID NO: 1203 is the sequence formulaC-X(22-24)-P-[PSR]-C-V-X(3)-R-C-X-G-C-C-X(6)-C-X(32-41)-C.

SEQ ID NO: 1204 is the sequence formula TNTFxxxP.

SEQ ID NO: 1205 is the sequence EFGVATNTFFKPPCVSVYRCG.

SEQ ID NO: 1206 is the sequence TNTFFKPP.

SEQ ID NO: 1207 is the sequence formula TNTFFKPPCVxxxR.

SEQ ID NO: 1208 is the sequence formula TNTFFKPPCVxxxRCGGCC

SEQ ID NO: 1209 is a sequence of a VEGF region involved in VEGFR-1binding.

SEQ ID NO: 1210 is a sequence of a VEGF-C region involved in VEGFR-3binding.

SEQ ID NO: 1211 is the sequence formula IEYIxxxS

SEQ ID NO: 1212 is the sequence formula TNTFX_(n)P

All publications and patents cited herein that are relevant to thedescription of the present invention are hereby incorporated byreference in their entirety.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those in the art. Accordingly, only such limitations as appearin the appended claims should be placed on the invention.

1-21. (canceled)
 22. A method of modulating the growth of mammalianendothelial cells or mammalian endothelial precursor cells, comprisingthe step of contacting the cells with a polypeptide, or a polynucleotidethat comprises a nucleotide sequence encoding the polypeptide, in anamount effective to modulate the growth of mammalian endothelial cells,wherein the polypeptide comprises an amino acid sequence at least 95%identical to a chimeric vascular endothelial growth factor amino acidsequence of the formula:NH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH wherein X₁ comprises an amino acidsequence selected from the group consisting of amino acids 3-11 of SEQID NO: 128 and amino acids 3-11 of SEQ ID NO: 137; wherein X₂ comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:129 and 138; wherein X₃ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 130 and 139; wherein X₄ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:131 and 140; wherein X₅ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 132 and 141; wherein X₆ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:133 and 142; wherein X₇ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 134 and 143; wherein X₈ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:135 and 144; wherein X₉ comprises an amino acid sequence selected fromthe group consisting of SE ID NOs: 136 and 145; whereinNH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH is not identical to amino acids 34to 135 of SEQ ID NO: 2 or amino acids 112 to 216 of SEQ ID NO: 22; andwherein the polypeptide binds to at least one receptor selected from thegroup consisting of human VEGFR-1, human VEGFR-2, and human VEGFR-3. 23.A method of modulating the growth of mammalian hematopoietic progenitorcells, comprising the step of contacting the cells with a polypeptide,or a polynucleotide that comprises a nucleotide sequence encoding thepolypeptide, in an amount effective to modulate the growth of mammalianendothelial cells, wherein the polypeptide comprises an amino acidsequence at least 95% identical to a chimeric vascular endothelialgrowth factor amino acid sequence of the formula:NH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH wherein X₁ comprises an amino acidsequence selected from the group consisting of amino acids 3-11 of SEQID NO: 128 and amino acids 3-11 of SEQ ID NO: 137; wherein X₂ comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:129 and 138; wherein X₃ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 130 and 139; wherein X₄ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:131 and 140; wherein X₅ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 132 and 141; wherein X₆ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:133 and 142; wherein X₇ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 134 and 143; wherein X₈ comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:135 and 144; wherein X₉ comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 136 and 145; whereinNH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH is not identical to amino acids 34to 135 of SEQ ID NO: 2 or amino acids 112 to 216 of SEQ ID NO: 22; andwherein the polypeptide binds to at least one receptor selected from thegroup consisting of human VEGFR-1, human VEGFR-2, and human VEGFR-3.24-89. (canceled)
 90. The method according to claim 22, wherein thecontacting comprises administering a composition comprising saidpolypeptide or said polynucleotide and a pharmaceutically acceptablecarrier to a mammalian subject.
 91. The method according to claim 23,wherein the contacting administering a composition comprising saidpolypeptide or said polynucleotide and a pharmaceutically acceptablecarrier to a mammalian subject.
 92. The method of claim 90, wherein thepolypeptide selectively stimulates a vascular endothelial receptorselected from the group consisting of VEGFR-1 and VEGFR-3.
 93. Themethod of claim 91, wherein the polypeptide selectively stimulates avascular endothelial receptor selected from the group consisting ofVEGFR-1 and VEGFR-3.
 94. The method according to claim 22 or 23, whereinthe polypeptide further includes a signal peptide amino acid sequenceconnected to the amino acid sequence of the formulaNH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₉-COOH.
 95. The method according to claim 22or 23, wherein the polypeptide further includes an amino-terminalmethionine residue.
 96. The method according to claim 22 or 23, whereinthe polypeptide further includes a tag amino acid sequence connected tothe amino acid sequence of the formulaNH₂-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-COOH.
 97. The method according to claim22 or 23, wherein the polypeptide further comprises additional flankingsequence from VEGF-A (SEQ ID NO: 2) or VEGF-C (SEQ ID NO: 22).
 98. Themethod according to claim 22 or 23, wherein the polypeptide furtherincludes one or more amino acid sequences selected from the groupconsisting of a prepro-VEGF-C signal peptide, a prepro-VEGF-Camino-terminal propeptide, and a prepro-VEGF-C carboxy-terminalpro-peptide.
 99. The method according to claim 22 or 23, wherein thepolypeptide binds to human VEGFR-1 and VEGFR-3.
 100. The methodaccording to claim 22 or 23, wherein the polypeptide binds to humanVEGFR-1, VEGFR-2, and VEGFR-3.
 101. The method according to claim 22 or23, wherein the polypeptide binds to exactly one of human VEGFR-1, humanVEGFR-2 and human VEGFR-3.
 102. The method according to claim 22 or 23,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of amino acids 1-102 as set forth in SEQ ID NO: 51;amino acids 1-102 as set forth in SEQ ID NO: 59; amino acids 1-102 asset forth in SEQ ID NO: 63; amino acids 1-104 as set forth in SEQ ID NO:67; amino acids 1-104 as set forth in SEQ ID NO: 71; amino acids 1-104as set forth in SEQ ID NO: 75; amino acids 1-104 as set forth in SEQ IDNO: 77; amino acids 1-104 as set forth in SEQ ID NO: 153; amino acids1-105 as set forth in SEQ ID NO: 155; amino acids 1-105 as set forth inSEQ ID NO: 157; amino acids 1-105 as set forth in SEQ ID NO: 159; aminoacids 1-103 as set forth in SEQ ID NO: 161; amino acids 1-102 as setforth in SEQ ID NO: 163; amino acids 1-104 as set forth in SEQ ID NO:165; amino acids 1-104 as set forth in SEQ ID NO: 167; amino acids 1-104as set forth in SEQ ID NO: 169; amino acids 1-104 as set forth in SEQ IDNO: 171; amino acids 1-104 as set forth in SEQ ID NO: 173; and aminoacids 1-104 as set forth in SEQ ID NO:
 175. 103. The method according toclaim 22 or 23, wherein the polypeptide comprises an amino acid sequenceat least 95% identical to the amino acid sequence of SEQ ID NO: 175,wherein said polypeptide binds at least one human receptor selected fromthe group consisting of VEGFR-1, VEGFR-2, and VEGFR-3.